Glycan therapeutics and method of treating conditions associated with TMAO

ABSTRACT

Preparations of glycan therapeutics, pharmaceutical compositions and medical foods thereof, optionally comprising micronutrients, polyphenols, prebiotics, probiotics, or other agents are provided and methods of making same. Also provided are methods of using said gycan therapeutics, e.g. for the modulation of human gastrointestinal microbiota and to treat dysbioses.

CLAIM OF PRIORITY

This application is a continuation of U.S. application Ser. No.16/395,929, filed Apr. 26, 2019, which is a continuation of U.S.application Ser. No. 16/126,873, filed Sep. 10, 2018, which is acontinuation of U.S. application Ser. No. 15/866,152, filed Jan. 9,2018, which is a continuation of U.S. Pat. No. 9,901,595, filed Oct. 5,2016, which is a continuation of U.S. Pat. No. 9,492,473, filed Feb. 5,2016, which is a continuation of International Application No.PCT/US2016/013305, filed Jan. 13, 2016, which claims priority to U.S.Application No. 62/108,039, filed Jan. 26, 2015; U.S. Application No.62/152,016, filed Apr. 23, 2015; U.S. Application No. 62/152,005, filedApr. 23, 2015; U.S. Application No. 62/152,007, filed Apr. 23, 2015;U.S. Application No. 62/152,011, filed Apr. 23, 2015; U.S. ApplicationNo. 62/152,017, filed Apr. 23, 2015; U.S. Application No. 62/216,995,filed Sep. 10, 2015; U.S. Application No. 62/216,997, filed Sep. 10,2015; U.S. Application No. 62/217,002, filed Sep. 10, 2015; U.S.Application No. 62/216,993, filed Sep. 10, 2015; U.S. Application No.62/238,112, filed Oct. 6, 2015; and U.S. Application No. 62/238,110,filed Oct. 6, 2015. The disclosure of each of the foregoing isincorporated herein by reference in its entirety.

BACKGROUND

The microbiota of humans is complex, and varies by individual dependingon genetics, age, sex, stress, nutrition and diet. The microbiotaperform many activities and may influence the physiology of the host.Changing the numbers and species of gut microbiota can alter communityfunction and interaction with the host. A limited number of probioticbacteria is known in the art, and some association with health benefitsdocumented when taken by humans. Some foods are considered ‘prebiotic’foods that contain substances that may promote the growth of certainbacteria that are thought to be beneficial to the human host. Theresults of clinical tests with these substances are conflicted withrespect to their efficacy, and their influence on human health isgenerally described as being modest. Thus, there is a need for noveltherapeutic inputs that can stimulate beneficial microbiota shifts andimprove human health.

SUMMARY OF THE INVENTION

In one aspect, the present invention features methods of modulating theabundance of a bacterial taxa in a human subject's gastrointestinalmicrobiota, the method comprising administering to the human subject apharmaceutical composition comprising a glycan therapeutic preparationin an amount effective to modulate the abundance of the bacterial taxa,wherein i) the glycan therapeutic preparation comprises a mixture ofbranched glycans, wherein the average degree of branching (DB, branchingpoints per residue) of the glycans in the preparation is at least 0.01(e.g., at least 0.05, or at least 0.1), ii) at least 50% of the glycansin the preparation have a degree of polymerization (DP) of at least 3and less than 30 glycan units, and iii) the ratio of alpha- tobeta-glycosidic bonds present in the glycans of the preparation isbetween about 1:1 to about 5:1 overall. In some embodiments, thebacterial taxa comprises at least a first and a second bacterial taxa.

In some embodiments, the preparation comprises branchedoligosaccharides. In some embodiments, the average degree of branchingin the preparation (DB) is at least 0.05 (e.g., at least 0.1).

In some embodiments, at least one, at least two, at least three, atleast four, or more of the glycosidic bonds independently comprise a 1→2glycosidic bond, a 1→3 glycosidic bond, a 1→4 glycosidic bond, or a 1→6glycosidic bond. In some embodiments, one or more, two or more, or threeor more glycosidic bonds are present in both alpha and betaconfiguration.

In some embodiments, a glycan unit comprises at least one, at least two,at least three, or more of a monosaccharide selected from the group of atetrose, a pentose, a hexose, and a heptose. In some embodiments, aglycan unit comprises at least one, at least two, at least three, ormore of a monosaccharide selected from the group of glucose, galactose,arabinose, mannose, fructose, xylose, fucose, and rhamnose.

In some embodiments, at least a plurality of the glycans, e.g., at least10, 20, 30 40, 50, 60, 70, 80, 90, 95, or 99% (by weight or number), orsubstantially all, of the glycans in the preparation, do not comprisemore than a preselected reference level, of a repeating unit of glycanunits, e.g., a repeating unit of 2, 3, 4 or more glycan units. In anembodiment, the preselected reference level is 10, 20, 30, 40, 50, or60% of the total glycan units in a glycan. By way of example, in anembodiment, a glycan made up of 20 saccharide monomers, less than 50% ofthose 20 monomers are repeating units of a 2 or 3 glycan repeat.

In some embodiments, the glycan therapeutic preparation is synthetic andnot isolated from a natural oligosaccharide or polysaccharide source.

In some embodiments, the abundance of the bacterial taxa (e.g., of eachof a first and a second bacterial taxa) in the human subject'sgastrointestinal microbiota is modulated by at least about 5%, 10%, 25%50%, 75%, 100%, 250%, 500%, 750%, or by at least 1000%. In someembodiments, the modulation comprises an increase or a decrease in theabundance of the bacterial taxa (e.g., of each of a first and a secondbacterial taxa) in the human subject's gastrointestinal microbiota.

In some embodiments, the bacterial taxa (e.g., a first and a secondbacterial taxa) comprises a commensal bacterial taxa. In otherembodiments, the bacterial taxa (e.g., a first and a second bacterialtaxa) comprises a pathogenic bacterial taxa.

In some embodiments, the bacterial taxa (e.g., a first and a secondbacterial taxa) comprises a genus selected from the group ofAkkermansia, Alistipes, Anaerofilum, Bacteroides, Bilophila, Blautia,Bifidobacterium, Butyrivibrio, Campylobacter, Candidatus, Citrobacter,Clostridium, Collinsella, Coprococcus, Desulfovibrio, Dialister, Dorea,Enterobacter, Enterococcus, Escherichia, Eubacterium, Faecalibacterium,Fusobacterium, Haemophilus, Klebsiella, Lachnospira, Lactobacillus,Odoribacter, Oscillospira, Parabacteroides, Peptococcus,Peptostreptococcus, Phascolarctobacterium, Porphyromonas, Portiera,Prevotella, Providencia, Pseudomonas, Roseburia, Ruminococcus,Salmonella, Shigella, Staphylococcus, Streptococcus, Subdoligranulum,Vibrio, and Yersinia. In some embodiments, the bacterial taxa (e.g., afirst and a second bacterial taxa) comprises a genus selected from thegroup of Prevotella, Akkermansia, Bacteroides, Clostridium(Erysipelotrichaceae), Clostridium (Clostridiaceae), Bifidobacterium,Aggregatibacter, Clostridium (Peptostreptococcaveae), Parabacteroides,Lactobacillus, and Enterococcus. In some embodiments, the bacterial taxa(e.g., a first and a second bacterial taxa) comprises a genus selectedfrom the group of Akkermansia, Bacteroides, Bifidobacterium,Lactobacillus, and Parabacteroides. In some embodiments, the bacterialtaxa (e.g., a first and a second bacterial taxa) comprises a genusselected from the group of Akkermansia and Blautia.

In some embodiments, the bacterial taxa (e.g., a first and a secondbacterial taxa) comprises a taxa predominant in the small intestine orlarge intestine. In some embodiments, the bacterial taxa (e.g., a firstand a second bacterial taxa) predominant in the small intestinecomprises a genus selected from the group of Achromobacter,Agrobacterium, Blautia, Burkholderia, Coprococcus, Cryocola,Enterococcus, Eubacterium, Holdemania, Lactococcus, Mycobacterium,Pseudoramibacter, Ralstonia, Sphingomonas, Streptococcus, andTuricibacter. In some embodiments, the bacterial taxa (e.g., a first anda second bacterial taxa) predominant in the large intestine comprises agenus selected from the group of Anaerotruncus, Akkermansia,Bacteroides, Bilophila, Butyricimonas, Odoribacter, Parabacteroides,Phascolarctobacterium, Prevotella, and Ruminococcus.

In some embodiments, the pharmaceutical composition further comprises apolyphenol preparation. In some embodiments, the polyphenol preparationcomprises a plant polyphenol isolated from a plant source material. Insome embodiments, the plant source material comprises blueberry,cranberry, grape, peach, plum, pomegranate, soy, red wine, black tea, orgreen tea.

In some embodiments, the modulating the abundance of a bacterial taxa(e.g., a first and a second bacterial taxa) treats a dysbiosis, e.g., adysbiosis described herein.

In another aspect, the present invention features a method of reducing adrug- or treatment-induced symptom in a human subject, comprisingadministering to the human subject a glycan therapeutic preparation inan amount effective to reduce a symptom induced by a drug or treatment,wherein i) the glycan therapeutic preparation comprises a mixture ofbranched glycans, wherein the average degree of branching (DB) of theglycans in the preparation is at least 0.01 (e.g., at least 0.05, or atleast 0.1), ii) at least 50% of the glycans in the preparation have adegree of polymerization (DP) of at least 3 and less than 30 glycanunits, and iii) the ratio of alpha- to beta-glycosidic bonds present inthe glycans of the preparation is between about 1:1 to about 5:1overall.

In some embodiments, the drug- or treatment-induced symptom is selectedfrom the group of bloating, diarrhea, vomiting, nausea, andconstipation. In some embodiments, the drug- or treatment-induced isdiarrhea. In some embodiments, the drug- or treatment-induced symptom isconstipation.

In some embodiments, the composition reduces drug-induced symptoms andthe composition is administered prior to, concomitant with, or afteradministration of the drug. In some embodiments, the drug is ananti-diabetic drug, an immune-suppressive drug, an antimicrobial drug, achemotherapeutic drug, an anti-psychotic drug, a proton pump inhibitordrug, or a non-steroid anti-inflammatory drug (NSAID). In someembodiments, the drug is selected from the group of ciprofloxacin,clindamycin, amoxicillin-clavulanate, cefixime, ephalosporins,fluoroquinolones, azithromycin, clarithromycin, erythromycin,tetracycline, azithromycin, irinotecan (camptosar), 5-fluorouracil,leucovorin, oxaliplatin, bortezomib, imatinib, lenalidomide, imbruvica,ipilimumab, pertuzumab, capecitabine, docetaxel, lapatinib, erlotinib,carmustine, etoposide, aracytine, melphalan, cytarabine, daunorubicine,amsacrine, mitoxantrone, olanzapine, ranitidine, famotidine, cimetidine,omeprazole, sucralfate, esomeprazole, naproxen, diclofenac,indomethacin, ibuprofen, ketoprofen, piroxicam, celecoxib, nimesulid,aspirin, metformin, paroxetine, valproic acid, and clozapine.

In some embodiments, the composition reduces treatment-induced symptomsand the treatment comprises radiation treatment or surgery.

In some embodiments, the drug- or treatment-induced symptom is exhibitedby the subject during a treatment regimen. In some embodiments, thereducing the one or more symptom increases compliance by the subject tothe treatment regimen. In some embodiments, the reducing the one or moresymptom increases the subject's tolerance to a higher dose of the drugto be administered during the treatment regimen.

In another aspect, the present invention features a method formodulating microbial diversity in a human subject's gastrointestinaltract, the composition comprising a glycan therapeutic preparation in anamount effective to modulate the microbial diversity, wherein i) theglycan therapeutic preparation comprises a mixture of branched glycans,wherein the average degree of branching (DB, branching points perresidue) of the glycans in the preparation is at least 0.01 (e.g., atleast 0.05, or at least 0.1), ii) at least 50% of the glycans in thepreparation have a degree of polymerization (DP) of at least 3 and lessthan 30 glycan units; and iii) the ratio of alpha- to beta-glycosidicbonds present in the glycans of the preparation is between about 1:1 toabout 5:1 overall. In some embodiments, the microbial diversitycomprises bacterial diversity. In some embodiments, the modulatingcomprises an increase or a decrease in microbial diversity.

In some embodiments, the microbial diversity is determined (e.g.,measured) by or expressed through use of the Shannon Diversity Index. Insome embodiments, the Shannon Diversity is increased or decreased by atleast about 5%. In some embodiments, the Shannon Diversity is increasedor decreased by at least about 15%. In some embodiments, the ShannonDiversity is increased or decreased by at least about 0.3 log-fold. Insome embodiments, Shannon Diversity is increased or decreased by atleast about 0.6 log-fold. In some embodiments, Shannon Diversity isincreased or decreased by at least about 1 log-fold.

In some embodiments, the abundance of at least one bacterial taxaselected from the group of genus Prevotella, Akkermansia, Bacteroides,Clostridium (Erysipelotrichaceae), Clostridium (Clostridiaceae),Bifidobacterium, Aggregatibacter, Clostridium (Peptostreptococcaveae),Parabacteroides, Lactobacillus, and Enterococcus is modulated. In someembodiments, the abundance of at least one bacterial taxa selected fromthe group of genus Prevotella, Akkermansia, Bacteroides, Clostridium(Erysipelotrichaceae), Clostridium (Clostridiaceae), Bifidobacterium,Aggregatibacter, Clostridium (Peptostreptococcaveae), Parabacteroides,Lactobacillus, and Enterococcus is increased by at least about 5%.

In some embodiments, the abundance of at least two bacterial taxaselected from the group of genus Prevotella, Akkermansia, Bacteroides,Clostridium (Erysipelotrichaceae), Clostridium (Clostridiaceae),Bifidobacterium, Aggregatibacter, Clostridium (Peptostreptococcaveae),Parabacteroides, Lactobacillus, and Enterococcus is modulated. In someembodiments, the abundance of at least two bacterial taxa selected fromthe group of genus Prevotella, Akkermansia, Bacteroides, Clostridium(Erysipelotrichaceae), Clostridium (Clostridiaceae), Bifidobacterium,Aggregatibacter, Clostridium (Peptostreptococcaveae), Parabacteroides,Lactobacillus, and Enterococcus is increased by at least about 5%.

In some embodiments, the abundance of at least one bacterial taxaselected from the group of genus Akkermansia, Bacteroides,Bifidobacterium, Lactobacillus, and Parabacteroides is modulated. Insome embodiments, the abundance of at least two bacterial taxa selectedfrom the group of genus Akkermansia, Bacteroides, Bifidobacterium,Lactobacillus, and Parabacteroides is modulated. In some embodiments,the abundance of at least one bacterial taxa selected from the group ofgenus Akkermansia and Blautia is modulated. In some embodiments, theabundance of both of the bacterial genera Akkermansia and Blautia ismodulated. In some embodiments, the modulating the microbial diversitytreats a dysbiosis.

In another aspect, the present invention features a method of treating ahuman subject in need thereof, the method comprising: a) identifying ahuman subject in need of treatment for dysbiosis, and b) administeringto the human subject a pharmaceutical composition comprising a glycantherapeutic preparation in an amount effective to treat the dysbiosis,wherein i) the glycan therapeutic preparation comprises a mixture ofbranched glycans, wherein the average degree of branching (DB) of theglycans in the preparation is at least 0.01 (e.g., at least 0.05, or atleast 0.1), ii) at least 50% of the glycans in the preparation have adegree of polymerization (DP) of at least 3 and less than 30 glycanunits, and iii) the ratio of alpha- to beta-glycosidic bonds present inthe glycans of the preparation is between about 1:1 to about 5:1overall.

In some embodiments, the human subject has an infectious disease,disorder or condition. In some embodiments, the infectious disease,disorder or condition is selected from the group of Clostridiumdifficile infection (CDI); Vancomycin-resistant enterococci (VRE)infection, infectious colitis, or C. difficile colitis. In someembodiments, the infectious disease, disorder or condition is diarrheaselected from the group of Clostridium difficile associated diarrhea(CDAD), antibiotic-associated diarrhea (AAD), antibiotic-induceddiarrhea, travelers' diarrhea (TD), pediatric diarrhea, and (acute)infectious diarrhea.

In some embodiments, the human subject has a metabolic disease, disorderor condition. In some embodiments, the metabolic disease, disorder orcondition is selected from the group of obesity, (insulin resistance)pre-diabetes, type II diabetes, high fasting blood sugar(hyperglycemia), and metabolic syndrome. In some embodiments, themetabolic disease, disorder or condition is a cardiovascular risk factorselected from the group of high blood cholesterol, high LDL, high bloodpressure (hypertension), high triglyceride levels, low HDL.

In some embodiments, the human subject has an inflammatory disease,disorder or condition. In some embodiments, the inflammatory disease,disorder or condition is selected from the group of inflammatory boweldisease (IBD), ulcerative colitis (UC), Crohn's disease (CD), intestinalinflammation, and microscopic colitis. In some embodiments, theinflammatory disease, disorder or condition is selected from the groupof irritable bowel syndrome (IBS), constipation, diarrhea, indigestion,and non-ulcer dyspepsia.

In some embodiments, the human subject has an autoimmune disease,disorder, or condition. In some embodiments, the autoimmune disease,disorder or condition is selected from the group of autoimmunearthritis, type I diabetes, multiple sclerosis, and psoriasis. In someembodiments, the human subject has an allergy. In some embodiments, theallergy comprises asthma or atopic dermatitis.

In some embodiments, the human subject has a neurological disease,disorder, or condition. In some embodiments, the neurological disease,disorder or condition is selected from the group of autism,hyperammonemia, and hepatic encephalopathy.

In some embodiments, treating further comprises administering a seconddrug or pharmaceutical agent. In some embodiments, the second drug orpharmaceutical agent is a standard-of-care drug or agent. In someembodiments, the treatment effects of the pharmaceutical compositioncomprising a glycan therapeutic preparation and the second drug orpharmaceutical agent are additive. In some embodiments, the treatmenteffects of the pharmaceutical composition comprising a glycantherapeutic preparation and the second drug or pharmaceutical agent aresynergistic.

In some embodiments, the composition is administered daily. In someembodiments, the composition is administered each day for apredetermined number of days (the treatment period). In someembodiments, the treatment period comprises between about 1 day andabout 30 days. In some embodiments, the treatment period comprisesbetween about 1 month and about 6 months. In some embodiments, thesubject is administered the composition for a single treatment period.In some embodiments, the subject is administered the composition formore than one treatment period.

In some embodiments, by treating the dysbiosis the disease of the humansubject is treated.

In another aspect, the present invention features a method of modulatinga functional pathway of the microbiota of a human subject'sgastrointestinal tract, the composition comprising a glycan therapeuticpreparation in an amount effective to modulate the functional pathway,wherein i) the glycan therapeutic preparation comprises a mixture ofbranched glycans, wherein the average degree of branching (DB) of theglycans in the preparation is at least 0.01 (e.g., at least 0.05, or atleast 0.1); ii) at least 50% of the glycans in the preparation have adegree of polymerization (DP) of at least 3 and less than 30 glycanunits; and iii) the ratio of alpha- to beta-glycosidic bonds present inthe glycans of the preparation is between about 1:1 to about 5:1overall.

In some embodiments, the functional pathway modulates the production ofan anti-microbial agent, a secondary bile acid, a short-chain fattyacid, a siderophore, or a metabolite listed in Table 2 by themicrobiota. In some embodiments, the antimicrobial agent comprises abacteriocin or hydrogen peroxide. In some embodiments, the short-chainfatty acid comprises formate, butyrate, acetate, propionate, orvalerate. In some embodiments, the metabolite comprises2-hydroxyisobutyrate, 3-hydroxyisovalerate, 3-methylcrotonylglycine,3-methylcrotonylglycine, allantoin, betaine, formate, mannitol, p-cresolglucuronide, phenylacetylglycine, sarcosine, taurine, acetic acid,acetylaldehyde, ascorbic acid, butanedione, butyric acid, deoxycholicacid, ethylphenyl sulfate, formic acid, indole, isobutyric acid,isovaleric acid, propionic acid, serotonin, succinic acid, succinate,TMAO, tryptophan, valeric acid, ursodeoxycholic acid, lactate, lacticacid, or hydrogen peroxide.

In some embodiments, the functional pathway modulates the level of aninflammatory or immunomodulatory cytokine in the human subject. In someembodiments, the inflammatory and immunomodulatory cytokine comprisesinterleukin-1α (IL-1α), IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12,IL-13, IL-17A, IL-17F, IL-22, IL-23, tumor necrosis factor (TNF),chemokine (C-C motif) ligand 5 (CCL5, also known as RANTES),transforming growth factor beta (TGF-β), or interferon gamma (IFN-γ).

In some embodiments, the functional pathway increases the level of ashort-chain fatty acid in the subject. In some embodiments, the increasein the short-chain fatty acid induces the generation of regulatory T(Treg) cells by the subject. In some embodiments, the increase in theshort-chain fatty acid reduces the permeability of the intestinal orplasma endotoxin level in the subject. In some embodiments, the increaseof a short-chain fatty acid reduces the inflammatory response of thesubject. In some embodiments, the short-chain fatty acid is produced byat least one bacterial species of the Ruminocaccaceae and/orLachnospiraceae family.

In some embodiments, the subject is obese.

In another aspect, the present invention features a method of preventinga relapse of a Clostridium difficile infection in a human subjectpreviously administered a drug for the treatment of a C. difficileinfection, the method comprising administering a glycan therapeuticpreparation in an amount effective to prevent the relapse, wherein i)the glycan therapeutic preparation comprises a mixture of branchedglycans, wherein the average degree of branching (DB) of the glycans inthe preparation is at least 0.01 (e.g., at least 0.05, or at least 0.1);ii) at least 50% of the glycans in the preparation have a degree ofpolymerization (DP) of at least 3 and less than 30 glycan units; andiii) the ratio of alpha- to beta-glycosidic bonds present in the glycansis between about 1:1 to about 5:1 overall.

In some embodiments, the relapse comprises the recurrence of one or moresymptoms associated with a C. difficile infection. In some embodiments,the relapse occurs during or after a first-line or standard of care drugtreatment regimen.

In some embodiments, the drug for the treatment of a C. difficileinfection is an antibiotic. In some embodiments, the antibiotic isselected from the group of vancomycin, metronidazole, and fidaxomicin.In some embodiments, the composition is administered concurrently orafter administration of the drug for the treatment of a C. difficileinfection.

In some embodiments, the composition is administered in combination witha second drug or treatment. In some embodiments, the second drug ortreatment comprises an antibiotic. In some embodiments, the antibioticis selected from the group of vancomycin, metronidazole, andfidaxomicin.

In some embodiments, administration of the composition results in areduction of the severity of a symptom associated with a C. difficileinfection in the subject but does not eliminate the population of C.difficile in the subject. In some embodiments, administration of thecomposition results in a reduction of the severity of a symptomassociated with a C. difficile infection in the subject but does notchange the level of the population of C. difficile in the subject.

In another aspect, the present invention features a method of making apharmaceutical composition, the method comprising a) providing apreparation comprising a mixture of synthetic glycans, b) acquiring avalue for one or more of the following characteristics of thepreparation, c) the degree of polymerization (DP), d) the average degreeof branching (DB), e) the ratio of alpha-glycosidic to beta-glycosidicbonds, and f) formulating the preparation as a pharmaceuticalcomposition if one or more of the following criteria are met: i) atleast 50% of the glycans in the preparation have a DP of at least 3 andless than 30 glycan units, ii) the average degree of branching (DB) ofthe glycans in the preparation is at least 0.01 (e.g., at least 0.05, orat least 0.1), iii) the ratio of alpha- to beta-glycosidic bonds presentin the glycans of the preparation is between about 1:1 to about 5:1overall.

In some embodiments, the method further comprises a) acquiring a valuefor any one or both additional characteristics of the preparation: i)the identity of the glycan units, ii) the ratio of glycan units, and b)formulating the preparation as a pharmaceutical composition if: iii) theglycan unit ratio in the preparation is about the same as the ratio ofthe glycan unit input.

In some embodiments, the method further comprises: b) acquiring a valuefor any one or both additional characteristics of the preparation: iv)the level of bacterial growth, in media supplemented with thepreparation, of commensal strains selected from the group consisting ofBacteroides caccae ATCC 43185, Prevotella copri DSM 18205, Bacteroidesthetaiotamicron ATCC 29741, Bacteroides cellulosilyticus DSM 14838,Clostridium scindens ATCC 35704, Ruminococcus obeum ATCC 29714,Clostridium nexile ATCC 27757, and Parabacteroides distasonis ATCC 8503,v) the level of bacterial growth, in media supplemented with thepreparation, of pathogenic strains selected from the group consisting ofClostridium difficile ATCC BAA-1382, Clostridium difficile ATCC 43255,Enterococcus faecium ATCC 700221, and Salmonella enterica ATCC 27869,and c) formulating the preparation as a pharmaceutical composition ifone or both of the following criteria are met: vi) promotion by themedia supplemented with the preparation of growth of at least 5commensal strains, vii) promotion by the media supplemented with thepreparation of growth of no more than 2 pathogenic strain. In someembodiments, step (b) may be performed prior to, concurrently with,another step in the process.

In some embodiments, the step of formulating the preparation as apharmaceutical composition comprises one or more of: i) removingunwanted constituents from the preparation, ii) reducing the volume ofthe preparation, iii) sterilizing the preparation, iv) admixing thepreparation with a pharmaceutically acceptable excipient or carrier, v)admixing the preparation with a second drug or pharmaceutical agent, vi)formulating the preparation into a aqueous solution or syrup, vii)formulating the preparation into a tablet or pill, and viii) formulatingthe preparation into a capsule.

In some embodiments, the step of formulating the preparation as apharmaceutical composition comprises one or more of ix) packaging thepreparation, x) labeling the packaged preparation, and xi) selling oroffering for sale the packaged and labeled preparation.

In another aspect, the present invention features a method of making apharmaceutical composition, the method comprising: (i) providing atherapeutic glycan preparation comprising at least one glycan unitselected from the group consisting of glucose, galactose, fucose,xylose, arabinose, rhamnose, and mannose, (ii) determining if apreselected NMR peak or group of NMR peaks is associated with the glycanpreparation, and (iii) if the preselected peak or group of peaks ispresent, formulating the preparation as a pharmaceutical composition.

In some embodiments, the peak is a 1H-13C HSQC NMR peak. In someembodiments, determining comprises acquiring a value for the identity ofan 1H-13C HSQC peak or group of peaks associated with the preparation,and if a preselected peak is present, formulating the preparation as apharmaceutical composition.

In some embodiments, i) for glycans comprising glucose, the peakscomprise at least one 1H-13C HSQC peak selected from 5.42, 92.5; 5.21,92.8; 5.18, 93.9; 5.08, 97.0; 5.36, 98.4; 5.34, 99.8; 5.38, 100.3; 4.95,98.6; 4.62, 96.6; 4.70, 103.6; 4.49, 103.4 1H shift (ppm) and 13C shift(ppm) or a corresponding peak; ii) for glycans comprising galactose, thepeaks comprise at least one 1H-13C HSQC peak selected from 5.37, 92.9;5.24, 93.1; 5.14, 96.0; 4.96, 99.3; 5.31, 98.7; 5.39, 101.4; 5.00,101.8; 4.80, 101.3; 4.63, 97.0; 4.56, 97.2; 4.53, 103.1; 4.43, 104.1 1Hshift (ppm) and 13C shift (ppm) or a corresponding peak; iii) forglycans comprising fucose, the peaks comprise at least one 1H-13C HSQCpeak selected from 5.18, 92.9; 5.33, 92.4; 5.04, 96.3; 4.90, 99.7; 4.52,97.0; 4.39, 103.6 1H shift (ppm) and 13C shift (ppm) or a correspondingpeak; iv) for glycans comprising xylose, the peaks comprise at least one1H-13C HSQC peak selected from 5.18, 93.0; 5.10, 94.3; 5.34, 98.2; 5.31,99.6; 5.11, 100.8; 4.91, 99.4; 4.56, 97.3; 4.64, 104.2; 4.54, 103.4;4.44, 102.6; 4.44, 104.1 1H shift (ppm) and 13C shift (ppm) or acorresponding peak; v) for glycans comprising arabinose, the peakscomprise at least one 1H-13C HSQC peak selected from 5.22, 93.2; 5.13,93.2; 5.29, 96.0; 5.26, 97.2; 5.12, 96.6; 5.18, 99.6; 5.06, 99.2; 4.99,100.0; 5.26, 101.9; 5.06, 102.1; 4.55, 97.4; 4.54, 105.2; 4.50, 105.5;4.38, 103.9 1H shift (ppm) and 13C shift (ppm) or a corresponding peak;vi) for glycans comprising rhamnose, the peaks comprise at least one1H-13C HSQC peak selected from 5.21, 93.2; 5.10, 94.5; 4.85, 94.1; 5.01,95.8; 5.35, 100.5; 5.15, 102.2; 5.04, 102.9; 4.78, 97.9; 4.71, 99.0;4.72, 101.0 1H shift (ppm) and 13C shift (ppm) or a corresponding peak;vii) for glycans comprising mannose, the peaks comprise at least one1H-13C HSQC peak selected from 5.37, 93.0; 5.16, 94.6; 4.88, 94.2; 5.39,101.7; 5.24, 101.9; 5.13, 102.8; 5.03, 102.7; 5.24, 105.6; 5.09, 108.0;4.88, 94.2; 4.89, 100.0; 4.70, 101.1 1H shift (ppm) and 13C shift (ppm)or a corresponding peak.

In some embodiments, i) for glycans comprising glucose, the peakscomprise at least two, at least three, at least four, or more 1H-13CHSQC peaks selected from 5.42, 92.5; 5.21, 92.8; 5.18, 93.9; 5.08, 97.0;5.36, 98.4; 5.34, 99.8; 5.38, 100.3; 4.95, 98.6; 4.62, 96.6; 4.70,103.6; 4.49, 103.4 1H shift (ppm) and 13C shift (ppm) or a correspondingpeak; ii) for glycans comprising galactose, the peaks comprise at leasttwo, at least three, at least four, or more 1H-13C HSQC peaks selectedfrom 5.37, 92.9; 5.24, 93.1; 5.14, 96.0; 4.96, 99.3; 5.31, 98.7; 5.39,101.4; 5.00, 101.8; 4.80, 101.3; 4.63, 97.0; 4.56, 97.2; 4.53, 103.1;4.43, 104.1 1H shift (ppm) and 13C shift (ppm) or a corresponding peak;iii) for glycans comprising fucose, the peaks comprise at least two, atleast three, at least four, or more 1H-13C HSQC peaks selected from5.18, 92.9; 5.33, 92.4; 5.04, 96.3; 4.90, 99.7; 4.52, 97.0; 4.39, 103.61H shift (ppm) and 13C shift (ppm) or a corresponding peak; iv) forglycans comprising xylose, the peaks comprise at least two, at leastthree, at least four, or more 1H-13C HSQC peaks selected from 5.18,93.0; 5.10, 94.3; 5.34, 98.2; 5.31, 99.6; 5.11, 100.8; 4.91, 99.4; 4.56,97.3; 4.64, 104.2; 4.54, 103.4; 4.44, 102.6; 4.44, 104.1 1H shift (ppm)and 13C shift (ppm) or a corresponding peak; v) for glycans comprisingarabinose, the peaks comprise at least two, at least three, at leastfour, or more 1H-13C HSQC peaks selected from 5.22, 93.2; 5.13, 93.2;5.29, 96.0; 5.26, 97.2; 5.12, 96.6; 5.18, 99.6; 5.06, 99.2; 4.99, 100.0;5.26, 101.9; 5.06, 102.1; 4.55, 97.4; 4.54, 105.2; 4.50, 105.5; 4.38,103.9 1H shift (ppm) and 13C shift (ppm) or a corresponding peak; vi)for glycans comprising rhamnose, the peaks comprise at least two, atleast three, at least four, or more 1H-13C HSQC peaks selected from5.21, 93.2; 5.10, 94.5; 4.85, 94.1; 5.01, 95.8; 5.35, 100.5; 5.15,102.2; 5.04, 102.9; 4.78, 97.9; 4.71, 99.0; 4.72, 101.0 1H shift (ppm)and 13C shift (ppm) or a corresponding peak; vii) for glycans comprisingmannose, the peaks comprise at least two, at least three, at least four,or more 1H-13C HSQC peaks selected from 5.37, 93.0; 5.16, 94.6; 4.88,94.2; 5.39, 101.7; 5.24, 101.9; 5.13, 102.8; 5.03, 102.7; 5.24, 105.6;5.09, 108.0; 4.88, 94.2; 4.89, 100.0; 4.70, 101.1 1H shift (ppm) and 13Cshift (ppm) or a corresponding peak.

In another aspect, the present invention features a pharmaceuticalcomposition comprising a therapeutic glycan preparation comprising amixture of branched glycans, wherein the average degree of branching(DB) is at least 0.01, wherein i) at least 50% of the glycans in thepreparation have a degree of polymerization (DP) of at least 3 and lessthan 30 glycan units, ii) the glycan preparation comprises both alpha-and beta-glycosidic bonds, iii) at least one of the glycosidic bondspresent in the glycans of the preparation comprise a 1→2 glycosidicbond, a 1→3 glycosidic bond, a 1→4 glycosidic bond, or a 1→6 glycosidicbond, and iv) the ratio of alpha- to beta-glycosidic bonds present inthe glycans of the preparation is between about 1:1 to about 5:1.

In some embodiments, at least one, at least two, at least three, atleast four, or more of the glycosidic bonds independently comprise a 1→2glycosidic bond, a 1→3 glycosidic bond, a 1→4 glycosidic bond, or a 1→6glycosidic bond. In some embodiments, one or more, two or more, or threeor more glycosidic bonds are present in both alpha and betaconfiguration.

In some embodiments, a glycan unit comprises at least one, at least two,at least three, or more of a monosaccharide selected from the group of atetrose, a pentose, a hexose, and a heptose. In some embodiments, aglycan unit comprises at least one, at least two, at least three, ormore of a monosaccharide selected from the group of glucose, galactose,arabinose, mannose, fructose, xylose, fucose, and rhamnose.

In some embodiments, at least a plurality of the glycans, e.g., at least10, 20, 30 40, 50, 60, 70, 80, 90, 95, or 99% (by weight or number), orsubstantially all, of the glycans in the preparation, do not comprisemore than a preselected reference level, of a repeating unit of glycanunits, e.g., a repeating unit of 2, 3, 4 or more glycan units. In anembodiment, the preselected reference level is 10, 20, 30, 40, 50, or60% of the total glycan units in a glycan. By way of example, in anembodiment, a glycan made up of 20 saccharide monomers, less than 50% ofthose 20 monomers are repeating units of a 2 or 3 glycan repeat.

In some embodiments, the glycan therapeutic preparation is synthetic andnot isolated from a natural oligosaccharide or polysaccharide source.

In some embodiments, the composition further comprises a polyphenolpreparation. In some embodiments, the composition further comprises apreparation of probiotic bacteria. In some embodiments, the compositionfurther comprises a drug or therapeutic agent. In some embodiments, thecomposition further comprises a pharmaceutically acceptable excipient.

In some embodiments, the composition is formulated as a unit-dosageform. In some embodiments, the unit-dosage form is formulated for oraldelivery. In some embodiments, the unit-dosage form is formulated todissolve in an aqueous solution and is orally administered as abeverage, syrup, solution, or suspension.

In some embodiments, the unit-dosage form is formulated as a delayedrelease or time controlled system. In some embodiments, the unit-dosageform is formulated to release the therapeutic glycan preparation in aspecific region of the GI tract. In some embodiments, the specificregion of the GI tract comprises the stomach, small intestine, largeintestine, or colon.

In some embodiments, the composition modulates the abundance of abacterial genus present in the GI tract. In some embodiments, thecomposition modulates the abundance of a bacterial genus present in oneor both of the small intestine or large intestine. In some embodiments,the composition modulates the abundance of a bacterial genus predominantin the small intestine selected from the group of genus Achromobacter,Agrobacterium, Blautia, Burkholderia, Coprococcus, Cryocola,Enterococcus, Eubacterium, Holdemania, Lactococcus, Mycobacterium,Pseudoramibacter, Ralstonia, Sphingomonas, Streptococcus, andTuricibacter. In some embodiments, the composition modulates theabundance of a bacterial genus predominant in the large intestineselected from the group of genus Anaerotruncus, Akkermansia,Bacteroides, Bilophila, Butyricimonas, Odoribacter, Parabacteroides,Phascolarctobacterium, Prevotella, and Ruminococcus.

In some embodiments, the unit-dosage form comprises about 0.1 mL toabout 5 mL of the therapeutic glycan preparation, is formulated for oraldelivery, and is formulated to release the therapeutic glycanpreparation in a specific region of the GI tract. In some embodiments,the unit-dosage form comprises about 0.1 mg to about 100 mg of thetherapeutic glycan preparation, is formulated for oral delivery, and isformulated to release the therapeutic glycan preparation in a specificregion of the GI tract.

In some embodiments, the unit-dosage form comprises about 0.1 mL toabout 5 mL of the therapeutic glycan preparation, is formulated for oraldelivery, and the composition modulates the abundance of a bacterialgenus selected from the group of Bacteroides, Butyricimonas,Odoribacter, Parabacteroides, Prevotella, Anaerotruncus,Phascolarctobacterium, Ruminococcus, Bilophila, Akkermansia, Cryocola,Mycobacterium, Enterococcus, Lactococcus, Streptococcus, Turicibacter,Blautia, Coprococcus, Holdemania, Pseudoramibacter, Eubacterium,Agrobacterium, Sphingomonas, Achromobacter, Burkholderia, and Ralstonia.

In some embodiments, the unit-dosage form comprises about 0.1 mg toabout 100 mg of the therapeutic glycan preparation, is formulated fororal delivery, and the composition modulates the abundance of abacterial genus selected from the group of Bacteroides, Butyricimonas,Odoribacter, Parabacteroides, Prevotella, Anaerotruncus,Phascolarctobacterium, Ruminococcus, Bilophila, Akkermansia, Cryocola,Mycobacterium, Enterococcus, Lactococcus, Streptococcus, Turicibacter,Blautia, Coprococcus, Holdemania, Pseudoramibacter, Eubacterium,Agrobacterium, Sphingomonas, Achromobacter, Burkholderia, and Ralstonia.

In some embodiments, the present invention features a pharmaceutical kitcomprising a) a glycan therapeutic preparation in an amount effective tomodulate the abundance of the bacterial taxa, wherein i) the glycantherapeutic preparation comprises a mixture of branched glycans, whereinthe average degree of branching (DB) of the glycans in the preparationis at least 0.01; ii) at least 50% of the glycans in the preparationhave a degree of polymerization (DP) of at least 3 and less than 30glycan units; and iii) the ratio of alpha- to beta-glycosidic bondspresent in the glycans of the preparation is between about 1:1 to about5:1, b) at least a second constituent selected from the group of apreparation of polyphenols, a preparation of probiotic bacteria, a drugor therapeutic agent, and a dietary component, c) instructionalmaterial, and d) packaging.

In another aspect, the present invention features a pharmaceuticalcomposition comprising a therapeutic glycan preparation comprising atleast one glycan unit selected from the group of: glucose, galactose,fucose, xylose, arabinose, rhamnose, and mannose, wherein thepreparation comprises a glycan unit associated with one or more of thefollowing 1H-13C HSQC peaks: i) for glycans comprising glucose, thepeaks comprise at least one of an 1H-13C HSQC peak selected from 5.42,92.5; 5.21, 92.8; 5.18, 93.9; 5.08, 97.0; 5.36, 98.4; 5.34, 99.8; 5.38,100.3; 4.95, 98.6; 4.62, 96.6; 4.70, 103.6; 4.49, 103.4 1H shift (ppm)and 13C shift (ppm) or a corresponding peak; ii) for glycans comprisinggalactose, the peaks comprise at least one of an 1H-13C HSQC peakselected from 5.37, 92.9; 5.24, 93.1; 5.14, 96.0; 4.96, 99.3; 5.31,98.7; 5.39, 101.4; 5.00, 101.8; 4.80, 101.3; 4.63, 97.0; 4.56, 97.2;4.53, 103.1; 4.43, 104.1 1H shift (ppm) and 13C shift (ppm) or acorresponding peak; iii) for glycans comprising fucose, the peakscomprise at least one of an 1H-13C HSQC peak selected from 5.18, 92.9;5.33, 92.4; 5.04, 96.3; 4.90, 99.7; 4.52, 97.0; 4.39, 103.6 1H shift(ppm) and 13C shift (ppm) or a corresponding peak; iv) for glycanscomprising xylose, the peaks comprise at least one of an 1H-13C HSQCpeak selected from 5.18, 93.0; 5.10, 94.3; 5.34, 98.2; 5.31, 99.6; 5.11,100.8; 4.91, 99.4; 4.56, 97.3; 4.64, 104.2; 4.54, 103.4; 4.44, 102.6;4.44, 104.1 1H shift (ppm) and 13C shift (ppm) or a corresponding peak;v) for glycans comprising arabinose, the peaks comprise at least one ofan 1H-13C HSQC peak selected from 5.22, 93.2; 5.13, 93.2; 5.29, 96.0;5.26, 97.2; 5.12, 96.6; 5.18, 99.6; 5.06, 99.2; 4.99, 100.0; 5.26,101.9; 5.06, 102.1; 4.55, 97.4; 4.54, 105.2; 4.50, 105.5; 4.38, 103.9 1Hshift (ppm) and 13C shift (ppm) or a corresponding peak; vi) for glycanscomprising rhamnose, the peaks comprise at least one of an 1H-13C HSQCpeak selected from 5.21, 93.2; 5.10, 94.5; 4.85, 94.1; 5.01, 95.8; 5.35,100.5; 5.15, 102.2; 5.04, 102.9; 4.78, 97.9; 4.71, 99.0; 4.72, 101.0 1Hshift (ppm) and 13C shift (ppm) or a corresponding peak; vii) forglycans comprising mannose, the peaks comprise at least one of an 1H-13CHSQC peak selected from 5.37, 93.0; 5.16, 94.6; 4.88, 94.2; 5.39, 101.7;5.24, 101.9; 5.13, 102.8; 5.03, 102.7; 5.24, 105.6; 5.09, 108.0; 4.88,94.2; 4.89, 100.0; 4.70, 101.1 1H shift (ppm) and 13C shift (ppm) or acorresponding peak.

In some embodiments, the pharmaceutical composition comprises atherapeutic glycan preparation comprising at least one glycan unitselected from the group of: glucose, galactose, fucose, xylose,arabinose, rhamnose, and mannose, wherein the preparation comprises aglycan unit associated with two or more of the following 1H-13C HSQCpeaks: i) for glycans comprising glucose, the peaks comprise at leasttwo, at least three, at least four, or more 1H-13C HSQC peaks selectedfrom 5.42, 92.5; 5.21, 92.8; 5.18, 93.9; 5.08, 97.0; 5.36, 98.4; 5.34,99.8; 5.38, 100.3; 4.95, 98.6; 4.62, 96.6; 4.70, 103.6; 4.49, 103.4 1Hshift (ppm) and 13C shift (ppm) or a corresponding peak; ii) for glycanscomprising galactose, the peaks comprise at least two, at least three,at least four, or more 1H-13C HSQC peaks selected from 5.37, 92.9; 5.24,93.1; 5.14, 96.0; 4.96, 99.3; 5.31, 98.7; 5.39, 101.4; 5.00, 101.8;4.80, 101.3; 4.63, 97.0; 4.56, 97.2; 4.53, 103.1; 4.43, 104.1 1H shift(ppm) and 13C shift (ppm) or a corresponding peak; iii) for glycanscomprising fucose, the peaks comprise at least two, at least three, atleast four, or more 1H-13C HSQC peaks selected from 5.18, 92.9; 5.33,92.4; 5.04, 96.3; 4.90, 99.7; 4.52, 97.0; 4.39, 103.6 1H shift (ppm) and13C shift (ppm) or a corresponding peak; iv) for glycans comprisingxylose, the peaks comprise at least two, at least three, at least four,or more 1H-13C HSQC peaks selected from 5.18, 93.0; 5.10, 94.3; 5.34,98.2; 5.31, 99.6; 5.11, 100.8; 4.91, 99.4; 4.56, 97.3; 4.64, 104.2;4.54, 103.4; 4.44, 102.6; 4.44, 104.1 1H shift (ppm) and 13C shift (ppm)or a corresponding peak; v) for glycans comprising arabinose, the peakscomprise at least two, at least three, at least four, or more 1H-13CHSQC peaks selected from 5.22, 93.2; 5.13, 93.2; 5.29, 96.0; 5.26, 97.2;5.12, 96.6; 5.18, 99.6; 5.06, 99.2; 4.99, 100.0; 5.26, 101.9; 5.06,102.1; 4.55, 97.4; 4.54, 105.2; 4.50, 105.5; 4.38, 103.9 1H shift (ppm)and 13C shift (ppm) or a corresponding peak; vi) for glycans comprisingrhamnose, the peaks comprise at least two, at least three, at leastfour, or more 1H-13C HSQC peaks selected from 5.21, 93.2; 5.10, 94.5;4.85, 94.1; 5.01, 95.8; 5.35, 100.5; 5.15, 102.2; 5.04, 102.9; 4.78,97.9; 4.71, 99.0; 4.72, 101.0 1H shift (ppm) and 13C shift (ppm) or acorresponding peak; vii) for glycans comprising mannose, the peakscomprise at least two, at least three, at least four, or more 1H-13CHSQC peaks selected from 5.37, 93.0; 5.16, 94.6; 4.88, 94.2; 5.39,101.7; 5.24, 101.9; 5.13, 102.8; 5.03, 102.7; 5.24, 105.6; 5.09, 108.0;4.88, 94.2; 4.89, 100.0; 4.70, 101.1 1H shift (ppm) and 13C shift (ppm)or a corresponding peak.

In another aspect, the present invention features a pharmaceuticalcomposition for use in modulating the abundance of a bacterial taxa in ahuman subject's gastrointestinal microbiota, the composition comprisinga glycan therapeutic preparation in an amount effective to modulate theabundance of the bacterial taxa, wherein i) the glycan therapeuticpreparation comprises a mixture of branched glycans, wherein the averagedegree of branching (DB, branching points per residue) of the glycans inthe preparation is at least 0.01 (e.g., at least 0.05, or at least 0.1),ii) at least 50% of the glycans in the preparation have a degree ofpolymerization (DP) of at least 3 and less than 30 glycan units, andiii) the ratio of alpha- to beta-glycosidic bonds present in the glycansof the preparation is between about 1:1 to about 5:1 overall. In someembodiments, the bacterial taxa comprises at least a first and a secondbacterial taxa.

In some embodiments, the preparation comprises branchedoligosaccharides. In some embodiments, the average degree of branchingin the preparation (DB) is at least 0.05 (e.g., at least 0.1).

In some embodiments, at least one, at least two, at least three, atleast four, or more of the glycosidic bonds independently comprise a 1→2glycosidic bond, a 1→3 glycosidic bond, a 1→4 glycosidic bond, or a 1→6glycosidic bond. In some embodiments, one or more, two or more, or threeor more glycosidic bonds are present in both alpha and betaconfiguration.

In some embodiments, a glycan unit comprises at least one, at least two,at least three, or more of a monosaccharide selected from the group of atetrose, a pentose, a hexose, and a heptose. In some embodiments, aglycan unit comprises at least one, at least two, at least three, ormore of a monosaccharide selected from the group of glucose, galactose,arabinose, mannose, fructose, xylose, fucose, and rhamnose.

In some embodiments, at least a plurality of the glycans, e.g., at least10, 20, 30 40, 50, 60, 70, 80, 90, 95, or 99% (by weight or number), orsubstantially all, of the glycans in the preparation, do not comprisemore than a preselected reference level, of a repeating unit of glycanunits, e.g., a repeating unit of 2, 3, 4 or more glycan units. In anembodiment, the preselected reference level is 10, 20, 30, 40, 50, or60% of the total glycan units in a glycan. By way of example, in anembodiment, a glycan made up of 20 saccharide monomers, less than 50% ofthose 20 monomers are repeating units of a 2 or 3 glycan repeat.

In some embodiments, the glycan therapeutic preparation is synthetic andnot isolated from a natural oligosaccharide or polysaccharide source.

In some embodiments, the abundance of the bacterial taxa (e.g., of eachof a first and a second bacterial taxa) in the human subject'sgastrointestinal microbiota is modulated by at least about 5%, 10%, 25%50%, 75%, 100%, 250%, 500%, 750%, or by at least 1000%. In someembodiments, the modulation comprises an increase or a decrease in theabundance of the bacterial taxa (e.g., of each of a first and a secondbacterial taxa) in the human subject's gastrointestinal microbiota.

In some embodiments, the bacterial taxa (e.g., a first and a secondbacterial taxa) comprises a commensal bacterial taxa. In otherembodiments, the bacterial taxa (e.g., a first and a second bacterialtaxa) comprises a pathogenic bacterial taxa.

In some embodiments, the bacterial taxa (e.g., a first and a secondbacterial taxa) comprises a genus selected from the group ofAkkermansia, Alistipes, Anaerofilum, Bacteroides, Bilophila, Blautia,Bifidobacterium, Butyrivibrio, Campylobacter, Candidatus, Citrobacter,Clostridium, Collinsella, Coprococcus, Desulfovibrio, Dialister, Dorea,Enterobacter, Enterococcus, Escherichia, Eubacterium, Faecalibacterium,Fusobacterium, Haemophilus, Klebsiella, Lachnospira, Lactobacillus,Odoribacter, Oscillospira, Parabacteroides, Peptococcus,Peptostreptococcus, Phascolarctobacterium, Porphyromonas, Portiera,Prevotella, Providencia, Pseudomonas, Roseburia, Ruminococcus,Salmonella, Shigella, Staphylococcus, Streptococcus, Subdoligranulum,Vibrio, and Yersinia. In some embodiments, the bacterial taxa (e.g., afirst and a second bacterial taxa) comprises a genus selected from thegroup of Prevotella, Akkermansia, Bacteroides, Clostridium(Erysipelotrichaceae), Clostridium (Clostridiaceae), Bifidobacterium,Aggregatibacter, Clostridium (Peptostreptococcaveae), Parabacteroides,Lactobacillus, and Enterococcus. In some embodiments, the bacterial taxa(e.g., a first and a second bacterial taxa) comprises a genus selectedfrom the group of Akkermansia, Bacteroides, Bifidobacterium,Lactobacillus, and Parabacteroides. In some embodiments, the bacterialtaxa (e.g., a first and a second bacterial taxa) comprises a genusselected from the group of Akkermansia and Blautia.

In some embodiments, the bacterial taxa (e.g., a first and a secondbacterial taxa) comprises a taxa predominant in the small intestine orlarge intestine. In some embodiments, the bacterial taxa (e.g., a firstand a second bacterial taxa) predominant in the small intestinecomprises a genus selected from the group of Achromobacter,Agrobacterium, Blautia, Burkholderia, Coprococcus, Cryocola,Enterococcus, Eubacterium, Holdemania, Lactococcus, Mycobacterium,Pseudoramibacter, Ralstonia, Sphingomonas, Streptococcus, andTuricibacter. In some embodiments, the bacterial taxa (e.g., a first anda second bacterial taxa) predominant in the large intestine comprises agenus selected from the group of Anaerotruncus, Akkermansia,Bacteroides, Bilophila, Butyricimonas, Odoribacter, Parabacteroides,Phascolarctobacterium, Prevotella, and Ruminococcus.

In some embodiments, the pharmaceutical composition further comprises apolyphenol preparation. In some embodiments, the polyphenol preparationcomprises a plant polyphenol isolated from a plant source material. Insome embodiments, the plant source material comprises blueberry,cranberry, grape, peach, plum, pomegranate, soy, red wine, black tea, orgreen tea.

In some embodiments, the modulating the abundance of a bacterial taxa(e.g., a first and a second bacterial taxa) treats a dysbiosis, e.g., adysbiosis described herein.

In another aspect, the present invention features a pharmaceuticalcomposition for use in reducing a drug- or treatment-induced symptom ina human subject, comprising administering to the human subject a glycantherapeutic preparation in an amount effective to reduce a symptominduced by a drug or treatment, wherein i) the glycan therapeuticpreparation comprises a mixture of branched glycans, wherein the averagedegree of branching (DB) of the glycans in the preparation is at least0.01 (e.g., at least 0.05, or at least 0.1), ii) at least 50% of theglycans in the preparation have a degree of polymerization (DP) of atleast 3 and less than 30 glycan units, and iii) the ratio of alpha- tobeta-glycosidic bonds present in the glycans of the preparation isbetween about 1:1 to about 5:1 overall.

In some embodiments, the drug- or treatment-induced symptom is selectedfrom the group of bloating, diarrhea, vomiting, nausea, andconstipation. In some embodiments, the drug- or treatment-induced isdiarrhea. In some embodiments, the drug- or treatment-induced symptom isconstipation.

In some embodiments, the composition reduces drug-induced symptoms andthe composition is administered prior to, concomitant with, or afteradministration of the drug. In some embodiments, the drug is ananti-diabetic drug, an immune-suppressive drug, an antimicrobial drug, achemotherapeutic drug, an anti-psychotic drug, a proton pump inhibitordrug, or a non-steroid anti-inflammatory drug (NSAID). In someembodiments, the drug is selected from the group of ciprofloxacin,clindamycin, amoxicillin-clavulanate, cefixime, ephalosporins,fluoroquinolones, azithromycin, clarithromycin, erythromycin,tetracycline, azithromycin, irinotecan (camptosar), 5-fluorouracil,leucovorin, oxaliplatin, bortezomib, imatinib, lenalidomide, imbruvica,ipilimumab, pertuzumab, capecitabine, docetaxel, lapatinib, erlotinib,carmustine, etoposide, aracytine, melphalan, cytarabine, daunorubicine,amsacrine, mitoxantrone, olanzapine, ranitidine, famotidine, cimetidine,omeprazole, sucralfate, esomeprazole, naproxen, diclofenac,indomethacin, ibuprofen, ketoprofen, piroxicam, celecoxib, nimesulid,aspirin, metformin, paroxetine, valproic acid, and clozapine.

In some embodiments, the composition reduces treatment-induced symptomsand the treatment comprises radiation treatment or surgery.

In some embodiments, the drug- or treatment-induced symptom is exhibitedby the subject during a treatment regimen. In some embodiments, thereducing the one or more symptom increases compliance by the subject tothe treatment regimen. In some embodiments, the reducing the one or moresymptom increases the subject's tolerance to a higher dose of the drugto be administered during the treatment regimen.

In another aspect, the present invention features a pharmaceuticalcomposition for use in modulating microbial diversity in a humansubject's gastrointestinal tract, the composition comprising a glycantherapeutic preparation in an amount effective to modulate the microbialdiversity, wherein i) the glycan therapeutic preparation comprises amixture of branched glycans, wherein the average degree of branching(DB, branching points per residue) of the glycans in the preparation isat least 0.01 (e.g., at least 0.05, or at least 0.1), ii) at least 50%of the glycans in the preparation have a degree of polymerization (DP)of at least 3 and less than 30 glycan units; and iii) the ratio ofalpha- to beta-glycosidic bonds present in the glycans of thepreparation is between about 1:1 to about 5:1 overall. In someembodiments, the microbial diversity comprises bacterial diversity. Insome embodiments, the modulating comprises an increase or a decrease inmicrobial diversity.

In some embodiments, the microbial diversity is determined (e.g.,measured) by or expressed through use of the Shannon Diversity Index. Insome embodiments, the Shannon Diversity is increased or decreased by atleast about 5%. In some embodiments, the Shannon Diversity is increasedor decreased by at least about 15%. In some embodiments, the ShannonDiversity is increased or decreased by at least about 0.3 log-fold. Insome embodiments, Shannon Diversity is increased or decreased by atleast about 0.6 log-fold. In some embodiments, Shannon Diversity isincreased or decreased by at least about 1 log-fold.

In some embodiments, the abundance of at least one bacterial taxaselected from the group of genus Prevotella, Akkermansia, Bacteroides,Clostridium (Erysipelotrichaceae), Clostridium (Clostridiaceae),Bifidobacterium, Aggregatibacter, Clostridium (Peptostreptococcaveae),Parabacteroides, Lactobacillus, and Enterococcus is modulated. In someembodiments, the abundance of at least one bacterial taxa selected fromthe group of genus Prevotella, Akkermansia, Bacteroides, Clostridium(Erysipelotrichaceae), Clostridium (Clostridiaceae), Bifidobacterium,Aggregatibacter, Clostridium (Peptostreptococcaveae), Parabacteroides,Lactobacillus, and Enterococcus is increased by at least about 5%.

In some embodiments, the abundance of at least two bacterial taxaselected from the group of genus Prevotella, Akkermansia, Bacteroides,Clostridium (Erysipelotrichaceae), Clostridium (Clostridiaceae),Bifidobacterium, Aggregatibacter, Clostridium (Peptostreptococcaveae),Parabacteroides, Lactobacillus, and Enterococcus is modulated. In someembodiments, the abundance of at least two bacterial taxa selected fromthe group of genus Prevotella, Akkermansia, Bacteroides, Clostridium(Erysipelotrichaceae), Clostridium (Clostridiaceae), Bifidobacterium,Aggregatibacter, Clostridium (Peptostreptococcaveae), Parabacteroides,Lactobacillus, and Enterococcus is increased by at least about 5%.

In some embodiments, the abundance of at least one bacterial taxaselected from the group of genus Akkermansia, Bacteroides,Bifidobacterium, Lactobacillus, and Parabacteroides is modulated. Insome embodiments, the abundance of at least two bacterial taxa selectedfrom the group of genus Akkermansia, Bacteroides, Bifidobacterium,Lactobacillus, and Parabacteroides is modulated. In some embodiments,the abundance of at least one bacterial taxa selected from the group ofgenus Akkermansia and Blautia is modulated. In some embodiments, theabundance of both of the bacterial genera Akkermansia and Blautia ismodulated. In some embodiments, the modulating the microbial diversitytreats a dysbiosis.

In another aspect, the present invention features a pharmaceuticalcomposition for use in treating a human subject in need thereof, thetreating comprising: a) identifying a human subject in need of treatmentfor dysbiosis, and b) administering to the human subject apharmaceutical composition comprising a glycan therapeutic preparationin an amount effective to treat the dysbiosis, wherein i) the glycantherapeutic preparation comprises a mixture of branched glycans, whereinthe average degree of branching (DB) of the glycans in the preparationis at least 0.01 (e.g., at least 0.05, or at least 0.1), ii) at least50% of the glycans in the preparation have a degree of polymerization(DP) of at least 3 and less than 30 glycan units, and iii) the ratio ofalpha- to beta-glycosidic bonds present in the glycans of thepreparation is between about 1:1 to about 5:1 overall.

In some embodiments, the human subject has an infectious disease,disorder or condition. In some embodiments, the infectious disease,disorder or condition is selected from the group of Clostridiumdifficile infection (CDI); Vancomycin-resistant enterococci (VRE)infection, infectious colitis, or C. difficile colitis. In someembodiments, the infectious disease, disorder or condition is diarrheaselected from the group of Clostridium difficile associated diarrhea(CDAD), antibiotic-associated diarrhea (AAD), antibiotic-induceddiarrhea, travelers' diarrhea (TD), pediatric diarrhea, and (acute)infectious diarrhea.

In some embodiments, the human subject has a metabolic disease, disorderor condition. In some embodiments, the metabolic disease, disorder orcondition is selected from the group of obesity, (insulin resistance)pre-diabetes, type II diabetes, high fasting blood sugar(hyperglycemia), and metabolic syndrome. In some embodiments, themetabolic disease, disorder or condition is a cardiovascular risk factorselected from the group of high blood cholesterol, high LDL, high bloodpressure (hypertension), high triglyceride levels, low HDL.

In some embodiments, the human subject has an inflammatory disease,disorder or condition. In some embodiments, the inflammatory disease,disorder or condition is selected from the group of inflammatory boweldisease (IBD), ulcerative colitis (UC), Crohn's disease (CD), intestinalinflammation, and microscopic colitis. In some embodiments, theinflammatory disease, disorder or condition is selected from the groupof irritable bowel syndrome (IBS), constipation, diarrhea, indigestion,and non-ulcer dyspepsia.

In some embodiments, the human subject has an autoimmune disease,disorder, or condition. In some embodiments, the autoimmune disease,disorder or condition is selected from the group of autoimmunearthritis, type I diabetes, multiple sclerosis, and psoriasis. In someembodiments, the human subject has an allergy. In some embodiments, theallergy comprises asthma or atopic dermatitis.

In some embodiments, the human subject has a neurological disease,disorder, or condition. In some embodiments, the neurological disease,disorder or condition is selected from the group of autism,hyperammonemia, and hepatic encephalopathy.

In some embodiments, treating further comprises administering a seconddrug or pharmaceutical agent. In some embodiments, the second drug orpharmaceutical agent is a standard-of-care drug or agent. In someembodiments, the treatment effects of the pharmaceutical compositioncomprising a glycan therapeutic preparation and the second drug orpharmaceutical agent are additive. In some embodiments, the treatmenteffects of the pharmaceutical composition comprising a glycantherapeutic preparation and the second drug or pharmaceutical agent aresynergistic.

In some embodiments, the composition is administered daily. In someembodiments, the composition is administered each day for apredetermined number of days (the treatment period). In someembodiments, the treatment period comprises between about 1 day andabout 30 days. In some embodiments, the treatment period comprisesbetween about 1 month and about 6 months. In some embodiments, thesubject is administered the composition for a single treatment period.In some embodiments, the subject is administered the composition formore than one treatment period.

In some embodiments, by treating the dysbiosis the disease of the humansubject is treated.

In another aspect, the present invention features a pharmaceuticalcomposition for use in modulating a functional pathway of the microbiotaof a human subject's gastrointestinal tract, the composition comprisinga glycan therapeutic preparation in an amount effective to modulate thefunctional pathway, wherein i) the glycan therapeutic preparationcomprises a mixture of branched glycans, wherein the average degree ofbranching (DB) of the glycans in the preparation is at least 0.01 (e.g.,at least 0.05, or at least 0.1); ii) at least 50% of the glycans in thepreparation have a degree of polymerization (DP) of at least 3 and lessthan 30 glycan units; and iii) the ratio of alpha- to beta-glycosidicbonds present in the glycans of the preparation is between about 1:1 toabout 5:1 overall.

In some embodiments, the functional pathway modulates the production ofan anti-microbial agent, a secondary bile acid, a short-chain fattyacid, a siderophore, or a metabolite listed in Table 2 by themicrobiota. In some embodiments, the antimicrobial agent comprises abacteriocin or hydrogen peroxide. In some embodiments, the short-chainfatty acid comprises formate, butyrate, acetate, propionate, orvalerate. In some embodiments, the metabolite comprises2-hydroxyisobutyrate, 3-hydroxyisovalerate, 3-methylcrotonylglycine,3-methylcrotonylglycine, allantoin, betaine, formate, mannitol, p-cresolglucuronide, phenylacetylglycine, sarcosine, taurine, acetic acid,acetylaldehyde, ascorbic acid, butanedione, butyric acid, deoxycholicacid, ethylphenyl sulfate, formic acid, indole, isobutyric acid,isovaleric acid, propionic acid, serotonin, succinic acid, succinate,TMAO, tryptophan, valeric acid, ursodeoxycholic acid, lactate, lacticacid, or hydrogen peroxide.

In some embodiments, the functional pathway modulates the level of aninflammatory or immunomodulatory cytokine in the human subject. In someembodiments, the inflammatory and immunomodulatory cytokine comprisesinterleukin-1α (IL-1α), IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12,IL-13, IL-17A, IL-17F, IL-22, IL-23, tumor necrosis factor (TNF),chemokine (C-C motif) ligand 5 (CCL5, also known as RANTES),transforming growth factor beta (TGF-β), or interferon gamma (IFN-γ).

In some embodiments, the functional pathway increases the level of ashort-chain fatty acid in the subject. In some embodiments, the increasein the short-chain fatty acid induces the generation of regulatory T(Treg) cells by the subject. In some embodiments, the increase in theshort-chain fatty acid reduces the permeability of the intestinal orplasma endotoxin level in the subject. In some embodiments, the increaseof a short-chain fatty acid reduces the inflammatory response of thesubject. In some embodiments, the short-chain fatty acid is produced byat least one bacterial species of the Ruminocaccaceae and/orLachnospiraceae family.

In some embodiments, the subject is obese.

In another aspect, the present invention features a pharmaceuticalcomposition for use in preventing a relapse of a Clostridium difficileinfection in a human subject previously administered a drug for thetreatment of a C. difficile infection, the composition comprising aglycan therapeutic preparation in an amount effective to prevent therelapse, wherein i) the glycan therapeutic preparation comprises amixture of branched glycans, wherein the average degree of branching(DB) of the glycans in the preparation is at least 0.01 (e.g., at least0.05, or at least 0.1); ii) at least 50% of the glycans in thepreparation have a degree of polymerization (DP) of at least 3 and lessthan 30 glycan units; and iii) the ratio of alpha- to beta-glycosidicbonds present in the glycans is between about 1:1 to about 5:1 overall.

In some embodiments, the relapse comprises the recurrence of one or moresymptoms associated with a C. difficile infection. In some embodiments,the relapse occurs during or after a first-line or standard of care drugtreatment regimen.

In some embodiments, the drug for the treatment of a C. difficileinfection is an antibiotic. In some embodiments, the antibiotic isselected from the group of vancomycin, metronidazole, and fidaxomicin.In some embodiments, the composition is administered concurrently orafter administration of the drug for the treatment of a C. difficileinfection.

In some embodiments, the composition is administered in combination witha second drug or treatment. In some embodiments, the second drug ortreatment comprises an antibiotic. In some embodiments, the antibioticis selected from the group of vancomycin, metronidazole, andfidaxomicin.

In some embodiments, administration of the composition results in areduction of the severity of a symptom associated with a C. difficileinfection in the subject but does not eliminate the population of C.difficile in the subject. In some embodiments, administration of thecomposition results in a reduction of the severity of a symptomassociated with a C. difficile infection in the subject but does notchange the level of the population of C. difficile in the subject

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A representative SEC curve between 16 and 20.5 minutes of aglu100 sample showing the average MW and the MW at 10% of maximumabsorption on both the leading and trailing edges of the curve.

FIG. 2. A representative anomeric region of an ¹H-¹³C HSQC spectrum of aglu100 sample showing the signal distribution of alpha- andbeta-glycosidic bonds

FIGS. 3A-3C. A representative anomeric region of an ¹H-¹³C HSQC spectrumof glu100 (FIG. 3A), glu50gal50 (FIG. 3B), and gal100 (FIG. 3C) samples,demonstrating the additive effect of the fingerprint peaks.

FIGS. 4A-4C, Representative GC chromatograms of three representativepermethylated and hydrolyzed glycans, glu50gal50 (FIG. 4A),man52glu29gal19 (FIG. 4B), and glu100 (FIG. 4C), showing distribution ofregiochemistry as assigned by comparison to known standards.

FIG. 5. A representative partial assignment of the peaks in the anomericregion of a glu100 sample ¹H-¹³C HSQC spectrum showing the separationbetween alpha and beta isomers in the ¹H axis, with alpha isomersdownfield (¹H>4.8 ppm in this case) and beta isomers upfield (¹H<4.8 ppmin this case). In addition, terminal and internal sugars can bedistinguished in the ¹³C axis with terminal sugars upfield (¹³C<94 ppmfor alpha and ¹³C<100 ppm for beta in this case) and internal sugarsdownfield (¹³C>94 ppm for alpha and ¹³C>100 ppm for beta in this case).

FIGS. 6A-6B. A portion of an exemplary catalyst with a polymericbackbone and side chains is illustrated in FIG. 6A. A portion of anexemplary catalyst, in which a side chain with the acidic group isconnected to the polymeric backbone by a linker and in which a sidechain with the cationic group is connected directly to the polymericbackbone is illustrated in FIG. 6B.

FIG. 7. Distances were calculated for each mouse between microbiotasampled at 1 day before and 5 days after glycan or water administratedas described in Example 10. The larger the distance, the bigger changein microbial composition is observed.

FIG. 8. Shannon diversity index. Paired Wilcoxon test was used tocalculate the significance of observed differences as described inExample 10.

FIGS. 9A-9B. Relative abundance of sequences assigned to genusAkkermansia, phylum Verrucomicrobia is shown in FIG. 9A. Relativeabundance of sequences assigned to genus Blautia, phylum Firmicutes isshown in FIG. 9B.

FIG. 10. Kaplan-Meier survival curve post C. difficile infection bytreatment group for all short treatment groups as described in Example12.

FIG. 11. Weight change 10 days post C. difficile infection for all shorttreatment groups (mean+/−s.e.) as described in Example 12.

FIG. 12. Change in relative abundance of bacteria by genus from directlybefore glycan treatment and C. difficile infection (Day −1) to directlyafter 6 days of glycan treatments and 4 days of Vancomycin treatment(Day 6) as described in Example 12. Only genera that change on average5% relative abundance are shown. Only 1 cage in the Water treatmentgroup had surviving animals on day 6.

FIG. 13. Predicted relative abundance of secondary bile acidbiosynthesis pathway on Day 6, directly following treatment with glycansor vancomycin as described in Example 12. Open circles represent cagesand black circles represent mean with +/−s.d. C. difficile infectionoccurred on Day 0 and only cages with animals that survived infectionare shown (n=4 cages on Day 0). (*P<0.05, Wilcoxon Rank Sum Test).

FIG. 14. Log fold-change in alpha diversity (as measured by Shannonindex) from Day −1, directly before glycan treatment in infection withC. difficile, to Day 6, following treatment with glycans or vancomycinas described in Example 12. Points represent alpha diversity of a singlecage and lines represent median alpha diversity.

FIG. 15. Percent weight change of mice compared to day 0 of study(mean+/−s.e.). 2.5% DSS was administered from days 0 to 5 in all groupsas described in Example 13. Acacia fiber, glu100, and man52glu29gal19was administered from days −7 to day 14 in treatment groups.

FIG. 16. Endoscopy score measuring colonic inflammation on day 14 of thestudy as described in Example 13. Horizontal bars represent medianendoscopy score. **P<0.01,*P<0.05; Wilcoxon Rank Sum Test withbonferroni correction for multiple hypotheses.

FIGS. 17A-17B. Slopes of % weight change from day 0 to day 41 in micetreated with glu100 (0.3%; FIG. 17A), man52glu29gal19 (1%; FIG. 17B) andwater (both plots) as described in Example 14. The slopes of the glu100-and man52glu29gal19-treated groups were significantly different than theslope of the water-treated mice (P<0.001; linear mixed-effects modelwith significant interaction between study day and weight change;Regression lines are shown with shading representing +/− standard errorof the slope). Individual animal % weight changes are plotted (trianglesor circles).

FIG. 18. Day 39 blood glucose levels in mice fed a high fat diet, lowfat diet, or high fat diet with glu100 (0.3%) or man52glu29gal19 (1%)treatment as described in Example 14. Upper and lower hinges on boxplotcorrespond to the first and third quartiles and upper and lower whiskerextend to the highest and lowest value that is within 1.5 times theinter-quartile range, or distance between the first and third quartiles.Mice were gavaged with 2 g/kg (at a dose rate of 5 mL/kg) glucose inwater and blood glucose levels assessed at 2 hours post dose. Units onthe y-axis are mg/dL glucose.

FIG. 19. Epididymal fat pad weight on day 41 as a percentage of totalbody weight in high fat-fed mice, low fat-fed mice and high fat-fed micetreated with man52glu29gal19 (1%), fos (0.3%, 1%), and glu100 (0.3%) asdescribed in Example 14.

DETAILED DESCRIPTION OF THE INVENTION

In humans the gastrointestinal microbiota is largely stable when thehost is in good health; however, the ecosystem of the gastrointestinalmicrobiota varies depending on host age, disease, including infectionswith pathogens, stress, diet, and pharmaceutical treatments and canenter a state of dysbiosis. The invention relates to preparations ofglycan therapeutics and pharmaceutical compositions thereof (and medicalfoods or dietary supplements thereof), and related methods, which havebeen found to be effective to treat dysbiosis. The preparations ofglycan therapeutics and pharmaceutical compositions described hereinsurprisingly have a therapeutic effect on a number of diseases,disorders or pathological conditions, which may be associated withdysbiosis. While not wishing to be bound by theory, it is believed thatpreparations of glycan therapeutics and pharmaceutical compositionsdescribed herein work by modulating microbial organisms within a humanhost's gastrointestinal (GI) tract to cause a desired physiologicaleffect, such as improving health, in the host. The glycan therapeuticscan be selectively digested by certain microbial constituents therebyinducing specific changes in the GI tract, both in the compositionand/or activity (e.g. function) of the microbiota that confer benefitsupon host well-being and health. The glycan therapeutics can act astailored, finely tuned modulators for the resident or acquiredmicrobiota, e.g., enhancing or restoring the growth of beneficialbacteria and/or suppressing the growth of pathogenic microbes ormicrobes that are associated with a disease or condition.

The glycan therapeutics described herein can mediate shifts in theabundance of important taxa of the gastrointestinal microbiota (andassociated functional or genomic shifts), and methods are provided bywhich glycan therapeutics can alter the composition or function of thegastrointestinal microbiota. In some embodiments, the microbial shiftsallow multiple important microbiota properties to be introduced,modulated, increased, decreased, or stimulated. In some embodiments, themodulation includes alterations in i) ecosystem resilience todisturbance (or dysbioses), ii) microbiota diversity, iii) metaboliteproduction, iv) pathobiont and pathogen colonization, and v) alteredeffects on host metabolic, immune, and other functions or anycombination thereof.

Described herein are methods, compositions, and kits useful for thetreatment and/or prevention of dysbiosis and diseases possiblyassociated with a dysbiosis of the gastrointestinal microbiota and/orthe reduction of symptoms thereof in a subject in need thereof, and forimproving overall health of the host. Further described herein aredosage forms for glycan therapeutics. In some embodiments, the dosageforms are formulated for specific delivery to specific regions of the GItract, such as, e.g., the small or large intestine. Administration ofthe pharmaceutical compositions, medical foods or dietary supplementscomprising preparations of glycan therapeutics may treat or preventdysiosis, e.g., conditions in which a beneficial bacterial microbiota isdisturbed and in which the microbiota exhibit a dysbiosis. In someembodiments, the disturbance can be ameliorated by the use of the glycantherapeutics described herein so that improved physiological growth andfunction of both the beneficial microbiota and the host can be achieved.Such treatment or prevention may occur directly, e.g., a glycantherapeutic described herein may cause displacement of a pathogenicmicrobe with a non-pathogenic microbe or increase the growth ofbeneficial or commensal microbes, or it may occur indirectly, e.g., aglycan therapeutic described herein may affect metabolism or otherfunctions of the microbiota, thus modulating host physiology, e.g.,through the effect of one or more downstream metabolic products.Administration of glycan therapeutics described herein may improve theoverall health of the host and may restore a healthy equilibrium in aselected niche, such as the GI tract, by influencing one or more membersof the microbial community.

Generation of Glycan Therapeutic Preparations

Preparations comprising a plurality of glycans such as oligo- andpolysaccharide mixtures can be generated using a non-enzymatic catalyst,e.g., the polymeric catalyst described in U.S. Pat. No. 8,466,242, whichis incorporated herein by reference in its entirety, or by othersuitable methods. Methods to prepare the polymeric and solid-supportedcatalysts described herein can be found in WO 2014/031956, which ishereby incorporated by reference herein. The glycans generated, e.g., byusing the catalyst, can be structurally much more diverse glycans thanthose produced by enzymatic reactions.

Provided are also methods for generating the preparations of glycans(e.g. oligo- or polysaccharide compounds) described herein, by: a)providing one or more mono- or disaccharide glycan unit, or acombination thereof, b) contacting the mono- or disaccharides with anyof the polymeric catalysts described herein and a suitable solvent (suchas, e.g. water or a non-aqueous solvent) for a period of time sufficientto produce a polymerized species population (with a desired averagedegree of polymerization); and c) isolating and/or recovering at least aportion of the polymerized glycan preparation.

In some embodiments, preparations of glycans (e.g. oligo- orpolysaccharides) are polymolecular. In some embodiments, preparations ofglycans (e.g. oligo- or polysaccharides) are polymolecular andpolydisperse. For example, the glycan therapeutic preparations comprisea mixture of distinct oligosaccharide species (e.g. of different degreeof polymerization and degree of branching and different alpha-to-betaglycosidic bond ratios). In some embodiments, the glycan therapeuticpreparations comprise a plurality of distinct species (e.g.oligosaccharides) and may consist of 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷,1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, or more species invarious proportions to each other. Herein described are the averageproperties of the glycan therapeutic preparations, such as degree ofpolymerization, degree of branching, alpha- and beta-glycosidic bondratios, etc.

In certain embodiments, the starting material (comprising the glycanunits) is contacted with a polymer catalyst under conditions thatpromote the formation of one or more glycosidic bond between glycanunits, thereby producing a preparation of glycans. In some embodiments,the glycan unit is a monosaccharide. Suitable polymer catalysts compriseacidic monomers and ionic monomers that are connected to form apolymeric backbone, wherein each acidic monomer has at least oneBronsted-Lowry acid, and each ionic monomer independently has at leastone nitrogen-containing cationic group or phosphorous-containingcationic group. In some embodiments, each acidic monomer of the polymercatalyst may have one Bronsted-Lowry acid, and optionally theBronsted-Lowry acids are distinct. In some embodiments, each ionicmonomer of the polymer catalyst has one nitrogen-containing cationicgroup or phosphorous-containing cationic group. In some embodiments, atleast one ionic monomer of the polymer catalyst has twonitrogen-containing cationic groups or phosphorous-containing cationicgroups. A schematic outlining the general functional groups is shown inFIGS. 6A and 6B.

In certain embodiments, synthesis of the glycans (e.g. oligo- orpolysaccharides) using the polymeric catalyst is carried out in anaqueous environment. One suitable aqueous solvent is water, which may beobtained from various sources. Generally, water sources with lowerconcentrations of ionic species are preferable, as such ionic speciesmay reduce the effectiveness of the polymeric catalyst. In someembodiments where the aqueous solvent is water, the water has less than10% of ionic species (e.g., salts of sodium, phosphorous, ammonium,magnesium).

Generally, the polymeric catalyst and the glycan units are introducedinto an interior chamber of a reactor, either concurrently orsequentially. Glycan (e.g. oligo- or polysaccharides) synthesis can beperformed in a batch process or a continuous process. For example, inone embodiment, glycan synthesis is performed in a batch process, wherethe contents of the reactor are continuously mixed or blended, and allor a substantial amount of the products of the reaction are removed(e.g. isolated and/or recovered). In one variation, glycan synthesis isperformed in a batch process, where the contents of the reactor areinitially intermingled or mixed but no further physical mixing isperformed. In another variation, glycan synthesis is performed in abatch process, wherein once further mixing of the contents, or periodicmixing of the contents of the reactor, is performed (e.g., at one ormore times per hour), all or a substantial amount of the products of thereaction are removed (e.g. isolated and/or recovered) after a certainperiod of time.

In other embodiments, glycan (e.g. oligo- or polysaccharide) synthesisis performed in a continuous process, where the contents flow throughthe reactor with an average continuous flow rate but with no explicitmixing. After introduction of the polymeric catalyst and glycan unitsinto the reactor, the contents of the reactor are continuously orperiodically mixed or blended, and after a period of time, less than allof the products of the reaction are removed (e.g. isolated and/orrecovered). In one variation, glycan synthesis is performed in acontinuous process, where the mixture containing the catalyst and glycanunits is not actively mixed. Additionally, mixing of catalyst and theglycan units may occur as a result of the redistribution of polymericcatalysts settling by gravity, or the non-active mixing that occurs asthe material flows through a continuous reactor.

In some embodiments of the method, the starting material for thepolymerization reaction is one or more glycan unit selected from one ormore monosaccharides, one or more disaccharides, or a combinationthereof. In some embodiments of the method, the starting material forthe polymerization reaction is one or more glycan unit selected from afuranose sugar and a pyranose sugar. In some embodiments of the method,the starting material for the polymerization reaction is one or moreglycan unit selected from a tetrose, a pentose, a hexose, or a heptose.In some embodiments of the method, the starting material for thepolymerization reaction is one or more glycan unit selected from aglucose, a galactose, an arabinose, a mannose, a fructose, a xylose, afucose, and a rhamnose, all optionally in either their L- or D-form, inalpha or beta configuration (for dimers), and/or a deoxy-form, whereapplicable, and any combination thereof. In some embodiments, the glycanunits are substituted or derivatized with one or more of an acetateester, sulfate half-ester, phosphate ester, or a pyruvyl cyclic acetalgroup, or have been otherwise derivatized at, e.g., at one or morehydroxyl groups.

The glycan units used in the methods described herein may include one ormore sugars. In some embodiments, the one or more sugars are selectedfrom monosaccharides, disaccharides, and trisaccharides, or any mixturesthereof. In some embodiments, the one or more sugars aremonosaccharides, such as one or more C5 or C6 monosaccharides. In someembodiments, the one or more sugars are C5 monosaccharides. In otherembodiments, the one or more sugars are C6 monosaccharides.

In some embodiments of the method, the starting material for thepolymerization reaction is one or more glycan unit selected from aminosugars, deoxy sugars, imino sugars, sugar acids, short-chained fattyacids, and sugar alcohols to produce hybrid glycans.

In some embodiments, the starting material for the polymerizationreaction is one or more glycan unit selected from monosaccharides andother carbohydrates including, but not limited to glycolaldehyde,glyceraldehyde, dihydroxyacetone, erythrose, threose, erythulose,arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose,galactose, glucose, gulose, idose, mannose, talose, fructose, psicose,sorbose, tagatose, fucose, fuculose, rhamnose, mannoheptulose,sedoheptulose, neuraminic acid, N-acetylneuraminic acid,N-acetylgalactosamine, N-acetylglucosamine, fructosamine, galactosamine,glucosamine, sorbitol, glycerol, erythritol, threitol, arabitol,xylitol, mannitol, sorbitol, galactitol, fucitol, and lactic acid.

In some embodiments, the starting material for the polymerizationreaction is one or more glycan unit selected from disaccharides andother carbohydrates including, but not limited to acarviosin,N-acetyllactosamine, allolactose, cellobiose, chitobiose,glactose-alpha-1,3-galactose, gentiobiose, isomalt, isomaltose,isomaltulose, kojibiose, lactitol, lactobionic acid, lactose, lactulose,laminaribiose, maltitol, maltose, mannobiose, melibiose, melibiulose,neohesperidose, nigerose, robinose, rutinose, sambubiose, sophorose,sucralose, sucrose, sucrose acetate isobutyrate, sucrose octaacetate,trehalose, turanose, vicianose, and xylobiose.

In some embodiments, the starting material for the polymerizationreaction is one or more glycan unit selected from an amino sugar, adeoxy sugar, an imino sugar, a sugar acid, a short-chained fatty acid,and a sugar alcohol.

In some embodiments, the glycan unit may exist as a salt (e.g., apharmaceutically acceptable salt), such as, e.g., a hydrochlorate,hydroiodate, hydrobromate, phosphate, sulfate, methanesulfate, acetate,formate, tartrate, malate, citrate, succinate, lactate, gluconate,pyruvate, fumarate, propionate, aspartate, glutamate, benzoate,ascorbate salt.

Suitable glycan units include amino sugars, such as, e.g. acarbose,N-acetylemannosamine, N-acetylmuramic acid, N-acetylneuraminic acid,N-acetyletalosaminuronic acid, arabinopyranosyl-N-methyl-N-nitrosourea,D-fructose-L-histidine, N-glycolyneuraminic acid, ketosamine, kidamycin,mannosamine, 1B-methylseleno-N-acetyl-D-galactosamine, muramic acid,muramyl dipeptide, phosphoribosylamine, PUGNAc, sialyl-Lewis A,sialyl-Lewis X, validamycin, voglibose, N-acetylgalactosamine,N-acetylglucosamine, aspartylglucosamine, bacillithiol, daunosamine,desosamine, fructosamine, galactosamine, glucosamine, meglumine, andperosamine.

Suitable glycan units include deoxy sugars, such as, e.g.1-5-ahydroglucitol, cladinose, colitose, 2-deoxy-D-glucose,3-deoxyglucasone, deoxyribose, dideoxynucleotide, digitalose,fludeooxyglucose, sarmentose, and sulfoquinovose.

Suitable glycan units include imino sugars, such as, e.g.castanospermine, 1-deoxynojirimycin, iminosugar, miglitol, miglustat,and swainsonine.

Suitable glycan units include sugar acids, such as, e.g.N-acetylneuraminic acid, N-acetyltalosamnuronic acid, aldaric acid,aldonic acid, 3-deoxy-D-manno-oct-2-ulosonic acid, glucuronic acid,glucosaminuronic acid, glyceric acid, N-glycolylneuraminic acid,iduronic acid, isosaccharinic acid, pangamic acid, sialic acid, threonicacid, ulosonic acid, uronic acid, xylonic acid, gluconic acid, ascorbicacid, ketodeoxyoctulosonic acid, galacturonic acid, galactosaminuronicacid, mannuronic acid, mannosaminuronic acid, tartaric acid, mucic acid,saccharic acid, lactic acid, oxalic acid, succinic acid, hexanoic acid,fumaric acid, maleic acid, butyric acid, citric acid, glucosaminic acid,malic acid, succinamic acid, sebacic acid, and capric acid.

Suitable glycan units include short chained fatty acids, such as, e.g.,formic acid, acetic acid, propionic acid, butryic acid, isobutyric acid,valeric acid, and isovaleric acid.

Suitable glycan units include sugar alcohols, such as, e.g., methanol,ethylene glycol, glycerol, erythritol, threitol, arabitol, ribitol,xylitol, mannitol, sorbitol, galactitol, iditol, volemitol, fucitol,inositol, maltotritol, maltotetraitol, and polyglycitol.

The glycan units (e.g. sugars) used in the methods described herein maybe obtained from any commercially known sources, or produced accordingto any methods known in the art.

Reaction Conditions

In some embodiments, the glycan unit and catalyst (e.g., polymericcatalyst or solid-supported catalyst) are allowed to react for at least1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6hours, at least 8 hours, at least 16 hours, at least 24 hours, at least36 hours, or at least 48 hours; or between 1-24 hours, between 2-12hours, between 3-6 hours, between 1-96 hours, between 12-72 hours, orbetween 12-48 hours.

In some embodiments, the degree of polymerization of the one or moreoligosaccharides produced according to the methods described herein canbe regulated by the reaction time. For example, in some embodiments, thedegree of polymerization of the one or more oligosaccharides isincreased by increasing the reaction time, while in other embodiments,the degree of polymerization of the one or more oligosaccharides isdecreased by decreasing the reaction time.

Reaction Temperature

In some embodiments, the reaction temperature is maintained in the rangeof about 25° C. to about 150° C. In certain embodiments, the temperatureis from about 30° C. to about 125° C., about 60° C. to about 120° C.,about 80° C. to about 115° C., about 90° C. to about 110° C., about 95°C. to about 105° C., or about 100° C. to 110° C.

Amount of Glycan Units

The amount of the glycan unit used in the methods described hereinrelative to the amount solvent used may affect the rate of reaction andyield. The amount of the glycan unit used may be characterized by thedry solids content. In certain embodiments, dry solids content refers tothe total solids of a slurry as a percentage on a dry weight basis. Insome embodiments, the dry solids content of the glycan unit is betweenabout 5 wt % to about 95 wt %, between about 10 wt % to about 80 wt %,between about 15 wt %, to about 75 wt %, or between about 15 wt %, toabout 50 wt %.

Amount of Catalyst

The amount of the catalyst used in the methods described herein maydepend on several factors including, for example, the selection of thetype of glycan unit, the concentration of the glycan unit, and thereaction conditions (e.g., temperature, time, and pH). In someembodiments, the weight ratio of the catalyst to the glycan unit isabout 0.01 g/g to about 50 g/g, about 0.01 g/g to about 5 g/g, about0.05 g/g to about 1.0 g/g, about 0.05 g/g to about 0.5 g/g, about 0.05g/g to about 0.2 g/g, or about 0.1 g/g to about 0.2 g/g.

Solvent

In certain embodiments, the methods of using the catalyst are carriedout in an aqueous environment. One suitable aqueous solvent is water,which may be obtained from various sources. Generally, water sourceswith lower concentrations of ionic species (e.g., salts of sodium,phosphorous, ammonium, or magnesium) are preferable, as such ionicspecies may reduce effectiveness of the catalyst. In some embodimentswhere the aqueous solvent is water, the water has a resistivity of atleast 0.1 megaohm-centimeters, of at least 1 megaohm-centimeters, of atleast 2 megaohm-centimeters, of at least 5 megaohm-centimeters, or of atleast 10 megaohm-centimeters.

Water Content

Moreover, as the dehydration reaction of the methods progresses, wateris produced with each coupling of the one or more glycan units. Incertain embodiments, the methods described herein may further includemonitoring the amount of water present in the reaction mixture and/orthe ratio of water to monomer or catalyst over a period of time. In someembodiments, the method further includes removing at least a portion ofwater produced in the reaction mixture (e.g., by removing at least aboutany of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or100%, such as by vacuum filtration). It should be understood, however,that the amount of water to monomer may be adjusted based on thereaction conditions and specific catalyst used.

Any method known in the art may be used to remove water in the reactionmixture, including, for example, by vacuum filtration, vacuumdistillation, heating, and/or evaporation. In some embodiments, themethod comprises including water in the reaction mixture.

In some aspects, provided herein are methods of producing anoligosaccharide composition, by: combining a glycan unit and a catalysthaving acidic and ionic moieties to form a reaction mixture, whereinwater is produced in the reaction mixture; and removing at least aportion of the water produced in the reaction mixture. In certainvariations, at least a portion of water is removed to maintain a watercontent in the reaction mixture of less than 99%, less than 90%, lessthan 80%, less than 70%, less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, less than 10%, less than 5%, or less than1% by weight.

In some embodiments, the degree of polymerization of the one or moreoligosaccharides produced according to the methods described herein canbe regulated by adjusting or controlling the concentration of waterpresent in the reaction mixture. For example, in some embodiments, thedegree of polymerization of the one or more oligosaccharides isincreased by decreasing the water concentration, while in otherembodiments, the degree of polymerization of the one or moreoligosaccharides is decreased by increasing the water concentration. Insome embodiments, the water content of the reaction is adjusted duringthe reaction to regulate the degree of polymerization of the one or moreoligosaccharides produced.

In one example, to a round bottom flask equipped with an overheadstirrer and a jacketed short-path condenser one or more mono-, dimer-,trimer or other oligosaccharides may be added along with 1-50% (1-10%,1-20%, 1-30%, 1-40%, 1-60%, 1-70%) by dry weight of one or more of thecatalysts described herein. Water or another compatible solvent (0.1-5equiv, 1-5 equiv, 1-4 equiv, 0.1-4 equiv) may be added to the drymixture and the slurry can be combined at slow speed (e.g. 10-100 rpm,50-200 rpm, 100-200 rpm) using a paddle sized to match the contours ofthe selected round bottom flask as closely as possible. The mixture isheated to 70-180° C. (70-160° C., 75-165° C., 80-160° C.) under 10-1000mbar vacuum pressure. The reaction may be stirred for 30 minutes to 6hours, constantly removing water from the reaction. Reaction progresscan be monitored by HPLC. The solid mass obtained by the process can bedissolved in a volume of water sufficient to create a solution ofapproximately 50 Brix (grams sugar per 100 g solution). Once dissolutionis complete, the solid catalyst can be removed by filtration and theoligomer solution can be concentrated to approximately 50-75 Brix, e.g.,by rotary evaporation. Optionally, an organic solvent can be used andwater immiscible solvents can be removed by biphasic extraction andwater miscible solvents can be removed, e.g., by rotary evaporationconcomitant to the concentration step.

Additional Processing Steps

Optionally, the preparation may undergo additional processing steps.Additional processing steps may include, for example, purificationsteps. Purification steps may include, for example, separation,dilution, concentration, filtration, desalting or ion-exchange,chromatographic separation, or decolorization, or any combinationthereof.

Decolorization

In some embodiments, the methods described herein further include adecolorization step. The one or more oligosaccharides produced mayundergo a decolorization step using any method known in the art,including, for example, treatment with an absorbent, activated carbon,chromatography (e.g., using ion exchange resin), hydrogenation, and/orfiltration (e.g., microfiltration).

In certain embodiments, the one or more oligosaccharides produced arecontacted with a color-absorbing material at a particular temperature,at a particular concentration, and/or for a particular duration of time.In some embodiments, the mass of the color absorbing species contactedwith the one or more oligosaccharides is less than 50% of the mass ofthe one or more oligosaccharides, less than 35% of the mass of the oneor more oligosaccharides, less than 20% of the mass of the one or moreoligosaccharides, less than 10% of the mass of the one or moreoligosaccharides, less than 5% of the mass of the one or moreoligosaccharides, less than 2% of the mass of the one or moreoligosaccharides, or less than 1% of the mass of the one or moreoligosaccharides.

In some embodiments, the one or more oligosaccharides are contacted witha color absorbing material. In certain embodiments, the one or moreoligosaccharides are contacted with a color absorbing material for lessthan 10 hours, less than 5 hours, less than 1 hour, or less than 30minutes. In a particular embodiment, the one or more oligosaccharidesare contacted with a color absorbing material for 1 hour.

In certain embodiments, the one or more oligosaccharides are contactedwith a color absorbing material at a temperature from 20 to 100 degreesCelsius, 30 to 80 degrees Celsius, 40 to 80 degrees Celsius, or 40 to 65degrees Celsius. In a particular embodiment, the one or moreoligosaccharides are contacted with a color absorbing material at atemperature of 50 degrees Celsius.

In certain embodiments, the color absorbing material is activatedcarbon. In one embodiment, the color absorbing material is powderedactivated carbon. In other embodiments, the color absorbing material isan ion exchange resin. In one embodiment, the color absorbing materialis a strong base cationic exchange resin in a chloride form. In anotherembodiment, the color absorbing material is cross-linked polystyrene. Inyet another embodiment, the color absorbing material is cross-linkedpolyacrylate. In certain embodiments, the color absorbing material isAmberlite FPA91, Amberlite FPA98, Dowex 22, Dowex Marathon MSA, or DowexOptipore SD-2.

Ion-Exchange/De-Salting (Demineralization)

In some embodiments, the one or more oligosaccharides produced arecontacted with a material to remove salts, minerals, and/or other ionicspecies. In certain embodiments, the one or more oligosaccharides areflowed through an anionic/cationic exchange column pair. In oneembodiment, the anionic exchange column contains a weak base exchangeresin in a hydroxide form and the cationic exchange column contains astrong acid exchange resin in a protonated form.

Separation and Concentration

In some embodiments, the methods described herein further includeisolating the one or more oligosaccharides produced. In certainvariations, isolating the one or more oligosaccharides comprisesseparating at least a portion of the one or more oligosaccharides fromat least a portion of the catalyst, using any method known in the art,including, for example, centrifugation, filtration (e.g., vacuumfiltration, membrane filtration), and gravity settling. In someembodiments, isolating the one or more oligosaccharides comprisesseparating at least a portion of the one or more oligosaccharides fromat least a portion of any unreacted sugar, using any method known in theart, including, for example, filtration (e.g., membrane filtration),chromatography (e.g., chromatographic fractionation), differentialsolubility, and centrifugation (e.g., differential centrifugation).

In some embodiments, the methods described herein further include aconcentration step. For example, in some embodiments, the isolatedoligosaccharides undergo evaporation (e.g., vacuum evaporation) toproduce a concentrated oligosaccharide composition. In otherembodiments, the isolated oligosaccharides undergo a spray drying stepto produce an oligosaccharide powder. In certain embodiments, theisolated oligosaccharides undergo both an evaporation step and a spraydrying step.

Water Content

Moreover, as the dehydration reaction of the methods progresses, wateris produced with each coupling of the one or more sugars. In certainembodiments, the methods described herein may further include monitoringthe amount of water present in the reaction mixture and/or the ratio ofwater to sugar or catalyst over a period of time. In some embodiments,the method further includes removing at least a portion of waterproduced in the reaction mixture (e.g., by removing at least about anyof 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or 100%,such as by vacuum filtration). It should be understood, however, thatthe amount of water to sugar may be adjusted based on the reactionconditions and specific catalyst used.

Any method known in the art may be used to remove water in the reactionmixture, including, for example, by vacuum filtration, vacuumdistillation, heating, and/or evaporation. In some embodiments, themethod comprises including water in the reaction mixture.

In some aspects, provided herein are methods of producing anoligosaccharide composition, by: combining a glycan unit and a catalysthaving acidic and ionic moieties to form a reaction mixture, whereinwater is produced in the reaction mixture; and removing at least aportion of the water produced in the reaction mixture. In certainvariations, at least a portion of water is removed to maintain a watercontent in the reaction mixture of less than 99%, less than 90%, lessthan 80%, less than 70%, less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, less than 10%, less than 5%, or less than1% by weight.

In some embodiments, the degree of polymerization of the one or moreoligosaccharides produced according to the methods described herein canbe regulated by adjusting or controlling the concentration of waterpresent in the reaction mixture. For example, in some embodiments, thedegree of polymerization of the one or more oligosaccharides isincreased by decreasing the water concentration, while in otherembodiments, the degree of polymerization of the one or moreoligosaccharides is decreased by increasing the water concentration. Insome embodiments, the water content of the reaction is adjusted duringthe reaction to regulate the degree of polymerization of the one or moreoligosaccharides produced.

Fractionation

In some embodiments, the methods described herein further include afractionation step. Oligo- or polysaccharides prepared and purified maybe subsequently separated by molecular weight using any method known inthe art, including, for example, high-performance liquid chromatography,adsorption/desorption (e.g. low-pressure activated carbonchromatography), or filtration (for example, ultrafiltration ordiafiltration). In certain embodiments, prepared and purified glycansare separated into pools representing 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 98%, or greater than 98% short (about DP1-2), medium (aboutDP3-10), long (about DP11-18), or very long (about DP>18) species.

In certain embodiments, prepared glycans are fractionated by adsorptiononto a carbonaceous material and subsequent desorption of fractions bywashing the material with mixtures of an organic solvent in water at aconcentration of 1%, 5%, 10%, 20%, 50%, or 100%. In one embodiment, theadsorption material is activated charcoal. In another embodiment, theadsorption material is a mixture of activated charcoal and a bulkingagent such as diatomaceous earth or Celite 545 in 5%, 10%, 20%, 30%,40%, or 50% portion by volume or weight.

In further embodiments, prepared glycans are separated by passagethrough a high-performance liquid chromatography system. In certainvariations, prepared glycans are separated by ion-affinitychromatography, hydrophilic interaction chromatography, orsize-exclusion chromatography including gel-permeation andgel-filtration.

In other embodiments, low molecular weight materials are removed byfiltration methods. In certain variations, low molecular weightmaterials may be removed by dialysis, ultrafiltration, diafiltration, ortangential flow filtration. In certain embodiments, the filtration isperformed in static dialysis tube apparatus. In other embodiments, thefiltration is performed in a dynamic flow filtration system. In otherembodiments, the filtration is performed in centrifugal force-drivenfiltration cartridges.

Characteristics of Glycan Therapeutic Preparations

The glycan therapeutics described herein may comprise oligosaccharidesand/or polysaccharides. In some embodiments, the glycan therapeuticscomprise homo-oligo- or polysaccharides (or homoglycans), wherein allthe monosaccharides in a polysaccharide are of the same type. Glycantherapeutics comprising homopolysaccharides can include monosaccharidesbonded together via a single or multiple glycosidic bond types.

In some embodiments, the glycan therapeutics comprise hetero-oligo- orpolysaccharides (or heteroglycans), wherein more than one type ofmonosaccharide is present. Glycan therapeutics comprisingheteropolysaccharides can include distinct types of monosaccharidesbonded together via a single or multiple glycosidic bond types.

Monosaccharides are the building blocks of disaccharides (such assucrose and lactose) and polysaccharides (such as cellulose and starch).The glycan therapeutics may comprise a single type of monosaccharide(referred to as homopolymers or homoglycans) or a mixture (referred toas heteropolymers or heteroglycans). An oligosaccharide is a saccharidepolymer containing a small number (typically two to nine) of glycanunits (in this case, monosaccharides).

For example, fructo-oligosaccharides (FOS), which are found in manyvegetables, consist of short chains of fructose molecules, some of whichare terminated with a glucose molecule. Galactooligosaccharides (GOS),which also occur naturally, consist of short chains of galactosemolecules. These compounds can be only partially digested by humans.Oligosaccharides are primarily produced from the breakdown of naturalpolymers such as starch or inulin, from direct extractions out ofnatural substances, such as soybean, or from chemical or enzymaticsyntheses.

Polysaccharides are polymeric carbohydrate molecules composed of longchains of glycan units bound together by linkages, such as, e.g.glycosidic linkages. Polysaccharides contain more than ten glycan units(in this case, monosaccharides). Naturally occurring polysaccharides mayhave a general formula of C_(x)(H₂O)_(y) where x is usually a largenumber, e.g. between 10 and 2500. In some embodiments, hydrolysis may beused to generate the constituent monosaccharides or oligosaccharidesthat are suitable to produce the glycans described herein. Glycan units,such as e.g. monosaccharides, may exist in many different forms, forexample, conformers, cyclic forms, acyclic forms, stereoisomers,tautomers, anomers, and isomers.

In some embodiments, glycan therapeutic preparations (e.g. oligo- orpolysaccharides) are created that are polydisperse, exhibiting a rangeof degrees of polymerization. Optionally, the preparations may befractionated, e.g. representing 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, or greater than 98% short (about DP1-2), medium (aboutDP3-10), long (about DP11-18), or very long (about DP>18) species.

In one embodiment, a polydisperse, fractionated glycan therapeuticpreparation is provided comprising at least 85%, 90%, or at least 95%medium-length species with a DP of about 3-10. In one embodiment, apolydisperse, fractionated glycan therapeutic preparation is providedcomprising at least 85%, 90%, or at least 95% long-length species with aDP of about 11-18. In one embodiment, a polydisperse, fractionatedglycan therapeutic preparation is provided comprising at least 85%, 90%,or at least 95% very long-length species with a DP of about 18-30. Insome embodiments, the medium, long and very long fractionatedpreparations comprise an alpha- to beta-glycosidic bond ratio from 0.8:1to 5:1 or from 1:1 to 4:1. In some embodiments, the fractionatedpreparations have an average degree of branching of between about 0.01and about 0.2 or between about 0.05 and 0.1.

In some embodiments, methods are provided using the disclosed polymericcatalyst to control the molecular weight distribution of the glycans.For example, a majority, e.g. about 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or about 97% of the glycan therapeutic preparation has a DP ofbetween 2 and 25, between 3 and 25, between 4 and 25, between 5 and 25,between 6 and 25, between 7 and 25, between 8 and 25, between 9 and 25,between 10 and 25, between 2 and 30, between 3 and 30, between 4 and 30,between 5 and 30, between 6 and 30, between 7 and 30, between 8 and 30,between 9 and 30, or between 10 and 30.

In one embodiment, the glycan therapeutic preparation has a degree ofpolymerization (DP) of at least 3 and less than 30 glycan units.

In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or about 97% of the glycan therapeutic preparation has a DP of at least5 and less than 30 glycan units. In some embodiments, about 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycantherapeutic preparation has a DP of at least 8 and less than 30 glycanunits. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or about 97% of the glycan therapeutic preparation has a DP ofat least 10 and less than 30 glycan units. In some embodiments, about55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycantherapeutic preparation has a DP of between 3, 4, 5, 6, 7, 8 and 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 glycan units. In some embodiments,about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of theglycan therapeutic preparation has a DP of between 10, 11, 12, 13, 14,15, 16, 17, 18, 19 and 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 glycanunits. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or about 97% of the glycan therapeutic preparation has a DP ofbetween 3, 4, 5, 6, 7, 8, 9, 10 and 20, 21, 22, 23, 24, 25, 26, 27, 28glycan units.

In some embodiments, the glycan therapeutic preparation has a degree ofpolymerization (DP) distribution after combining the one or more glycanunits (e.g. sugars) with the polymeric catalyst (e.g., at 2, 3, 4, 8,12, 24, or 48 hours after combining the one or more glycan units (e.g.sugars) with the catalyst) is: DP2=0%-40%, such as less than 40%, lessthan 30%, less than 20%, less than 10%, less than 5%, or less than 2%;or 10%-30% or 15%-25%; DP3=0%-20%, such as less than 15%, less than 10%,less than 5%; or 5%-15%; and DP4+=greater than 15%, greater than 20%,greater than 30%, greater than 40%, greater than 50%; or 15%-75%,20%-40% or 25%-35%.

The yield of conversion for the one or more glycan units (e.g. sugars)in the methods described herein can be determined by any suitable methodknown in the art, including, for example, high performance liquidchromatography (HPLC). In some embodiments, the yield of conversion to aglycan therapeutic preparation with DP>1 after combining the one or moreglycan units with the catalyst (e.g., at 2, 3, 4, 8, 12, 24, or 48 hoursafter combining the one or more glycan units with the catalyst) isgreater than about 50% (e.g., greater than about 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 98%). In some embodiments, the yield ofconversion to a glycan therapeutic preparation with >DP2 after combiningthe one or more glycan units with the catalyst (e.g., at 2, 3, 4, 8, 12,24, or 48 hours after combining the one or more glycan units with thecatalyst) is greater than 30% (e.g., greater than 35%, 40%, 45%, 50%,55%. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%).

In one embodiment, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, orabout 97% of the glycan therapeutic preparation has a DP of at least 2.In one embodiment, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, orabout 97% of the glycan therapeutic preparation has a DP of at least 3.

In some embodiments, glycan therapeutic preparations are provided,wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.8%, or atleast 99.9% or even 100% of the glycan therapeutic preparation has adegree of polymerization (DP) of at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or at least 12 glycan units and less than 75, 70, 65, 60, 55, 50,45, 40, 35, 30, 25, 20, 19, 18, 17, 16, or less than 15 glycan units.

In some embodiments, glycan therapeutic preparations are provided,wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.8%, or atleast 99.9% or even 100% of the glycan therapeutic preparation has adegree of polymerization (DP) of at least 5 and less than 30 glycanunits, at least 8 and less than 30 glycan units.

In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or about 97% of the glycan therapeutic preparation has an average degreeof polymerization (DP) of about DP5, DP6, DP7, DP8, DP9, DP10, DP11, orDP12.

In some embodiments, glycan therapeutic preparations are providedwherein at least 50%, 60%, 70%, or 80% of the glycan therapeuticpreparation has a degree of polymerization of at least 3 and less than30 glycan units, or of at least 5 and less than 25 glycan units. In someembodiments, the average DP of the glycan therapeutic preparation isbetween about DP7 and DP9 or between about DP6 and DP10. In someembodiments, these glycan therapeutic preparations comprise an alpha- tobeta-glycosidic bond ratio from 0.8:1 to 5:1 or from 1:1 to 4:1. In someembodiments, the fractionated preparations have an average degree ofbranching of between about 0.01 and about 0.2 or between about 0.05 and0.1.

In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or about 97% of the glycan therapeutic preparation has an averagemolecular weight of about 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500,1550, 1600, 1650, 1700, 1750, 1800 g/mol and less than 1900, 2000, 2100,2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300,3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500,4600, 4700, 4800, 4900, and 5000 g/mol.

In some embodiments, the glycan preparations (e.g. oligo- orpolysaccharides) range in structure from linear to highly branched.Unbranched glycans may contain only alpha linkages or only betalinkages. Unbranched glycans may contain at least one alpha and at leastone beta linkage. Branched glycans may contain at least one glycan unitbeing linked via an alpha or a beta glycosidic bond so as to form abranch. The branching rate or degree of branching (DB) may vary, suchthat about every 2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th), 8^(th),9^(th), 10^(th), 15^(th), 20^(th), 25^(th), 30^(th), 35^(th), 40^(th),45^(th), 50^(th), 60^(th), or 70^(th) unit comprises at least onebranching point. For example, animal glycogen contains a branching pointapproximately every 10 units.

In some embodiments, preparations of glycan therapeutics are provided,wherein the preparation comprises a mixture of branched glycans, whereinthe average degree of branching (DB, branching points per residue) is 0,0.01. 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.99, 1, or 2. In some embodiments,preparations of glycan therapeutics are provided, wherein the averagedegree of branching is at least 0.01, 0.05, 0.1, 0.2, 0.3, or at least0.4. In some embodiments, preparations of glycan therapeutics areprovided, wherein the average degree of branching is between about 0.01and 0.1, 0.01 and 0.2, 0.01 and 0.3, 0.01 and 0.4, or 0.01 and 0.5. Insome embodiments, preparations of glycan therapeutics are provided,wherein the average degree of branching is not 0. In some embodiments,preparations of glycan therapeutics are provided, wherein the averagedegree of branching is not between at least 0.1 and less than 0.4 or atleast 0.2 and less than 0.4. In some embodiments, the preparations ofglycan therapeutics comprise linear glycans. In some embodiments, thepreparations of glycan therapeutics comprise glycans that exhibit abranched or branch-on-branch structure.

In some embodiments, preparations of glycan therapeutics are providedwherein the average degree of branching (DB) is not 0, but is at least0.01, 0.05, 0.1, or at least 0.2, or ranges between about 0.01 and about0.2 or between about 0.05 and 0.1.

Some glycans comprise oligosaccharides which have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. In accordance with accepted nomenclature, mostoligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right. Most oligosaccharides describedherein are described with the name or abbreviation for the non-reducingsaccharide (e.g., Gal or D-Gal), preceded or followed by theconfiguration of the glycosidic bond (alpha or beta), the ring bond, thering position of the reducing saccharide involved in the bond, and thenthe name or abbreviation of the reducing saccharide (e.g., Glc orD-Glc). The linkage (e.g., glycosidic linkage, galactosidic linkage,glucosidic linkage, etc.) between two sugar units can be expressed, forexample, as 1,4, 1→4, or (1-4), used interchangeably herein. Eachsaccharide can be in the cyclic form (e.g. pyranose or furanose form).For example, lactose is a disaccharide composed of cyclic forms ofgalactose and glucose joined by a beta (1-4) linkage where the acetaloxygen bridge is in the beta orientation.

Linkages between the individual glycan units found in preparations ofglycan therapeutics may include alpha 1→2, alpha 1→3, alpha 1→4, alpha1→6, alpha 2→1, alpha 2→3, alpha 2→4, alpha 2→6, beta 1→2, beta 1→3,beta 1→4, beta 1→6, beta 2→1, beta 2→3, beta 2→4, and beta 2→6.

In some embodiments, the glycan therapeutic preparations comprise onlyalpha linkages. In some embodiments, the glycan therapeutics compriseonly beta linkages. In some embodiments, the glycan therapeuticscomprise mixtures of alpha and beta linkages. In some embodiments, thealpha:beta glycosidic bond ratio in a preparation is about 0.1:1, 0.2:1,0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.2:1, 1.5:1,1.7:1, 2:1, 2.2:1, 2.5:1, 2.7:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, orabout 10:1.

In some embodiments, the glycan therapeutic preparation comprises bothalpha- and beta-glycosidic bonds selected from the group consisting of1→2 glycosidic bond, a 1→3 glycosidic bond, a 1→4 glycosidic bond, a 1→5glycosidic bond and a 1→6 glycosidic bond. In some embodiments, theglycan therapeutic preparation comprises at least two or at least threealpha and beta 1→2 glycosidic bonds, alpha and beta 1→3 glycosidicbonds, alpha and beta 1→4 glycosidic bonds, alpha and beta 1→5glycosidic bonds, and/or alpha and beta 1→6 glycosidic bonds. In someembodiments, the glycan therapeutic preparations comprise and alpha:betaglycosidic bond ratio in a preparation of about 0.8:1, 1:1, 2:1, 3:1,4:1 or 5:1, or it ranges from about 0.8:1 to about 5:1 or from about 1:1to about 4:1.

In some embodiments, the preparations of glycan therapeutics (e.g.oligosaccharides and polysaccharides) comprises a desired mixture ofglycan units with alpha- or beta configuration, e.g. the preparation ofglycan therapeutics comprises a desired ratio, such as: 1:1, 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1:25,1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85,1:90, 1:100, 1:150 of alpha- to beta-configuration or beta- toalpha-configuration.

In some embodiments, the preparations of glycan therapeutics (e.g.oligosaccharides and polysaccharides) comprises substantially all alpha-or beta configured glycan units, optionally comprising about 1%, 2%, 3%,4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,or 20% of the respective other configuration.

In some embodiments, the preparations of glycan therapeutics comprise atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, at least 99.9% or even 100% glycans with alpha glycosidic bonds. Insome embodiments, the preparations of glycan therapeutics comprise atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, at least 99.9% or even 100% glycans with beta glycosidic bonds. Insome embodiments, preparations of glycan therapeutics are provided,wherein at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, or at least 85% of glycans with glycosidic bondsthat are alpha glycosidic bonds, at least 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or at least 85% of glycanswith glycosidic bonds that are beta glycosidic bonds, and wherein thepercentage of alpha and beta glycosidic bonds does not exceed 100%.

In some embodiments, preparations of glycan therapeutics are provided,wherein at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, at least 99.9% or even 100% of glycan glycosidic bondsare one or more of: 1→2 glycosidic bonds, 1→3 glycosidic bonds, 1→4glycosidic bonds, and 1→6 glycosidic bonds. In some embodiments,preparations of glycan therapeutics are provided, wherein at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, at least 20%, or 25% each ofglycan glycosidic bonds are 1→2, 1→3, 1→4, and 1→6 glycosidic bonds.Optionally, the preparations of glycan therapeutics further comprise atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or at least 85% of glycanglycosidic bonds that are selected from the group consisting of: alpha2→1, alpha 2→3, alpha 2→4, alpha 2→6, beta 2→1, beta 2→3, beta 2→4, andbeta 2→6, glycosidic bonds.

In some embodiments, the preparations of glycan therapeutics compriseglycans with at least two glycosidic bonds selected from the groupconsisting of alpha 1→2 and alpha 1→3, alpha 1→2 and alpha 1→4, alpha1→2 and alpha 1→6, alpha 1→2 and beta 1→2, alpha 1→2 and beta 1→3, alpha1→2 and beta 1→4, alpha 1→2 and beta 1→6, alpha 1→3 and alpha 1→4, alpha1→3 and alpha 1→6, alpha 1→3 and beta 1→2, alpha 1→3 and beta 1→3, alpha1→3 and beta 1→4, alpha 1→3 and beta 1→6, alpha 1→4 and alpha 1→6, alpha1→4 and beta 1→2, alpha 1→4 and beta 1→3, alpha 1→4 and beta 1→4, alpha1→4 and beta 1→6, alpha 1→6 and beta 1→2, alpha 1→6 and beta 1→3, alpha1→6 and beta 1→4, alpha 1→6 and beta 1→6, beta 1→2 and beta 1→3, beta1→2 and beta 1→4, beta 1→2 and beta 1→6, beta 1→3 and beta 1→4, beta 1→3and beta 1→6, and beta 1→4 and beta 1→6.

For preparations comprising branched glycan therapeutics (e.g. thosewith a DB of 0.01. 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.99, 1, or 2) comprisinga side chain, which can be the same or a different side chain, the sidechain may be attached via one or more beta and alpha linkages, such as(1-2), (1-3), (1-4), (1-6), (2-3), (2-6) or other suitable linkages tothe main chain.

In some embodiments, preparations of glycan therapeutics are provided,wherein at least one glycan unit is a sugar in L-form. In someembodiments, preparations of glycans are provided, wherein at least oneglycan unit is a sugar in D-form. In some embodiments, preparations ofglycans are provided, wherein the glycan units are sugars in L- orD-form as they naturally occur or are more common (e.g. D-glucose,D-xylose, L-arabinose).

In some embodiments, the preparation of glycan therapeutics (e.g.oligosaccharides and polysaccharides) comprises a desired mixture of L-and D-forms of glycan units, e.g. of a desired ratio, such as: 1:1, 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20,1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80,1:85, 1:90, 1:100, 1:150 L- to D-forms or D- to L-forms.

In some embodiments, the preparation of glycan therapeutics comprisesglycans with substantially all L- or D-forms of glycan units, optionallycomprising about 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, or 20% of the respective other form.

In some embodiments, preparations of glycan therapeutics are provided,wherein at least one glycan unit is a tetrose, a pentose, a hexose, or aheptose. Optionally, the glycan units involved in the formation of theglycans (e.g. a mixture of branched oligosaccharides or polysaccharides)of the glycan therapeutic preparation are varied. Examples ofmonosaccharide glycan units include hexoses, such as glucose, galactose,and fructose, and pentoses, such as xylose. Monosaccharides generallyhave the chemical formula: C_(x)(H₂O)_(y), where conventionally x≥3.Monosaccharides can be classified by the number x of carbon atoms theycontain, for example: diose (2) triose (3) tetrose (4), pentose (5),hexose (6), and heptose (7). The monosaccharide glycan units may existin an acyclic (open-chain) form. Open-chain monosaccharides with samemolecular graph may exist as two or more stereoisomers. Themonosaccharides may also exist in a cyclic form through a nucleophilicaddition reaction between the carbonyl group and one of the hydroxyls ofthe same molecule. The reaction creates a ring of carbon atoms closed byone bridging oxygen atom. In these cyclic forms, the ring usually has 5(furanoses) or 6 atoms (pyranoses).

In some embodiments, the preparation of glycan therapeutics (e.g.oligosaccharides and polysaccharides) comprises a desired mixture ofdifferent monosaccharide glycan units, such as a mixture of a diose (2),a triose (3), tetrose (4), pentose (5), hexose (6), or heptose (7), inany desired ratio, e.g. for any two glycan units: 1:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1:25, 1:30,1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90,1:100, 1:150, etc., for any three glycan units:1:1:1, 1:2:1, 1:3:1,1:4:1, 1:5:1, 1:6:1, 1:7:1, 1:8:1, 1:9:1, 1:10:1, 1:12:1, 1:14:1,1:16:1, 1:18:1, 1:20:1, 1:1:2, 1:2:2, 1:3:2, 1:4:2, 1:5:2, 1:6:2, 1:7:2,1:8:2, 1:9:2, 1:10:2, 1:1:3, 1:2:3, 1:3:3, 1:4:3, 1:5:3, 1:6:3, 1:7:3,1:8:3, 1:9:3, 1:10:3, 1:1:4, 1:2:4, 1:3:4, 1:4:4, 1:5:4, 1:6:4, 1:7:4,1:8:4, 1:9:4, 1:10:4, 1:1:5, 1:2:5, 1:3:5, 1:4:5, 1:5:5, 1:6:5, 1:7:5,1:8:5, 1:9:5, 1:10:5, etc., for any four glycan units: 1:1:1:1, 1:2:2:1,1:3:2:1, 1:4:2:1, 1:5:2:1, 1:6:2:1, 1:7:2:1, 1:8:2:1, 1:9:2:1, 1:10:2:1,1:1:1:2, 1:2:2:2, 1:3:2:2, 1:4:2:2, 1:5:2:2, 1:6:2:2, 1:7:2:2, 1:8:2:2,1:9:2:2, 1:10:2:2, etc., for any five glycan units: 1:1:1:1:1,1:2:2:1:1, etc., for any six glycan units: 1:1:1:1:1:1, 1:1:1:1:1:2,etc., for any seven glycan units: 1:1:1:1:1:1:1, 1:1:1:1:1:1:2, etc.,and so on.

In some embodiments, the preparation of glycan therapeutics comprises adesired mixture of two, three, four or five different glycan units, suchas a mixture of, e.g., i) one or more glycan units selected frommonosaccharides, selected from glucose, a galactose, an arabinose, amannose, a fructose, a xylose, a fucose, and a rhamnose; ii) one or moreglycan units selected from disaccharides selected from acarviosin,n-acetyllactosamine, allolactose, cellobiose, chitobiose,glactose-alpha-1,3-galactose, gentiobiose, isomalt, isomaltose,isomaltulose, kojibiose, lactitol, lactobionic acid, lactose, lactulose,laminaribiose, maltitol, maltose, mannobiose, melibiose, melibiulose,neohesperidose, nigerose, robinose, rutinose, sambubiose, sophorose,sucralose, sucrose, sucrose acetate isobutyrate, sucrose octaacetate,trehalose, turanose, vicianose, and xylobiose; iii) one or more glycanunits selected from amino sugars selected from acarbose,N-acetylemannosamine, N-acetylmuramic acid, N-acetylnueraminic acid,N-acetyletalosaminuronic acid, arabinopyranosyl-N-methyl-N-nitrosourea,D-fructose-L-histidine, N-glycolyneuraminic acid, ketosamine, kidamycin,mannosamine, 1B-methylseleno-N-acetyl-D-galactosamine, muramic acid,muramyl dipeptide, phosphoribosylamine, PUGNAc, sialyl-Lewis A,sialyl-Lewis X, validamycin, voglibose, N-acetylgalactosamine,N-acetylglucosamine, aspartylglucosamine, bacillithiol, daunosamine,desosamine, fructosamine, galactosamine, glucosamine, meglumine, andperosamine; iv) one or more glycan units selected from deoxy sugarsselected from 1-5-ahydroglucitol, cladinose, colitose,2-deoxy-D-glucose, 3-deoxyglucasone, deoxyribose, dideoxynucleotide,digitalose, fludeooxyglucose, sarmentose, and sulfoquinovose; v) one ormore glycan units selected from imino sugars selected fromcastanospermine, 1-deoxynojirimycin, iminosugar, miglitol, miglustat,and swainsonine; one or more glycan units selected from sugar acidsselected from N-acetylneuraminic acid, N-acetyltalosamnuronic acid,aldaric acid, aldonic acid, 3-deoxy-D-manno-oct-2-ulosonic acid,glucuronic acid, glucosaminuronic acid, glyceric acid,N-glycolylneuraminic acid, iduronic acid, isosaccharinic acid, pangamicacid, sialic acid, threonic acid, ulosonic acid, uronic acid, xylonicacid, gluconic acid, ascorbic acid, ketodeoxyoctulosonic acid,galacturonic acid, galactosaminuronic acid, mannuronic acid,mannosaminuronic acid, tartaric acid, mucic acid, saccharic acid, lacticacid, oxalic acid, succinic acid, hexanoic acid, fumaric acid, maleicacid, butyric acid, citric acid, glucosaminic acid, malic acid,succinamic acid, sebacic acid, and capric acid; vi) one or more glycanunits selected from short-chain fatty acids selected from formic acid,acetic acid, propionic acid, butryic acid, isobutyric acid, valericacid, and isovaleric acid; and vii) one or more glycan units selectedfrom sugar alcohols selected from methanol, ethylene glycol, glycerol,erythritol, threitol, arabitol, ribitol, xylitol, mannitol, sorbitol,galactitol, iditol, volemitol, fucitol, inositol, maltotritol,maltotetraitol, and polyglycitol.

In some embodiments, the preparation of glycan therapeutics comprises aglycan unit or plurality of glycan units present in a salt form (e.g., apharmaceutically acceptable salt form), such as, e.g., a hydrochlorate,hydroiodate, hydrobromate, phosphate, sulfate, methanesulfate, acetate,formate, tartrate, malate, citrate, succinate, lactate, gluconate,pyruvate, fumarate, propionate, aspartate, glutamate, benzoate,ascorbate salt.

Exemplary glycans are described by a three-letter code representing themonomeric sugar component followed by a number out of one hundredreflecting the percentage of the material that monomer constitutes.Thus, ‘glu100’ is ascribed to a glycan generated from a 100% D-glucose(glycan unit) input and ‘glu50gal50’ is ascribed to a glycan generatedfrom 50% D-glucose and 50% D-galactose (glycan units) input or,alternatively from a lactose dimer (glycan unit) input. As used herein:xyl=D-xylose; ara=L-arabinose; gal=D-galactose; glu=D-glucose;rha=L-rhamnose; fuc=L-fucose; man=D-mannose; sor=D-sorbitol;gly=D-glycerol; neu=NAc-neuraminic acid.

In some embodiments, the preparation of glycan therapeutics comprisesone glycan unit A selected from i) to vii) above, wherein glycan unit Acomprises 100% of the glycan unit input. For example, in someembodiments, the glycan therapeutic preparation is selected from thehomo-glycans xyl100, rha100, ara100, gal100, glu100, and man100. In someembodiments, the glycan therapeutic preparation is selected from thehomo-glycans fuc100 and fru100.

In some embodiments, the preparation of glycan therapeutics comprises amixture of two glycan units A and B selected independently from i) tovii) above, wherein A and B may be selected from the same or a differentgroup i) to vii) and wherein A and B may be selected in any desiredratio (e.g. anywhere from 1-99% A and 99-1% B, not exceeding 100%).

For example, in some embodiments, the glycan therapeutic preparation isselected from the hetero-glycans ara50gal50, xyl75gal25, ara80xyl20,ara60xyl40, ara50xyl50, glu80man20, glu60man40, man60glu40, man80glu20,gal75xyl25, glu50gal50, man62glu38, and the hybrid glycans glu90sor10and glu90gly10.

In some embodiments, the preparation of glycan therapeutics comprises amixture of three glycan units A, B and C selected independently from i)to vii) above, wherein A, B and C may be selected from the same or adifferent group i) to vii) and wherein A, B and C may be selected in anydesired ratio (e.g. anywhere from 1-99% A, 1-99% B, 1-99% C, notexceeding 100%).

For example, in some embodiments, the glycan therapeutic preparation isselected from the hetero-glycans xyl75glu12gal12, xyl33glu33gal33,glu33gal33fuc33, man52glu29gal19, and the hybrid glycan glu33gal33neu33.

In some embodiments, the preparation of glycan therapeutics comprises amixture of four glycan units A, B, C and D selected independently fromi) to vii) above, wherein A, B, C and D may be selected from the same ora different group i) to vii) and wherein A, B, C and D may be selectedin any desired ratio (e.g. anywhere from 1-99% A, 1-99% B, 1-99% C,1-99% D, not exceeding 100%).

In some embodiments, the preparation of glycan therapeutics comprises amixture of five glycan units A, B, C, D and E selected independentlyfrom i) to vii) above, wherein A, B, C, D and E may be selected from thesame or a different group i) to vii) and wherein A, B, C, D and E may beselected in any desired ratio (e.g. anywhere from 1-99% A, 1-99% B,1-99% C, 1-99% D, 1-99% E, not exceeding 100%).

In some embodiments, preparations of glycan therapeutics are provided,wherein at least one glycan unit is selected from the group consistingof a glucose, a galactose, an arabinose, a mannose, a fructose, axylose, a fucose, and a rhamnose.

In some embodiments, the preparation of glycan therapeutics (e.g.oligosaccharides and polysaccharides) comprises a desired mixture of twodifferent monosaccharide glycan units, such as a mixture of, e.g.,glucose and galactose, glucose and arabinose, glucose and mannose,glucose and fructose, glucose and xylose, glucose and fucose, glucoseand rhamnose, galactose and arabinose, galactose and mannose, galactoseand fructose, galactose and xylose, galactose and fucose, and galactoseand rhamnose, arabinose and mannose, arabinose and fructose, arabinoseand xylose, arabinose and fucose, and arabinose and rhamnose, mannoseand fructose, mannose and xylose, mannose and fucose, and mannose andrhamnose, fructose and xylose, fructose and fucose, and fructose andrhamnose, xylose and fucose, xylose and rhamnose, and fucose andrhamnose, e.g. a in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8,1:9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45,1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, or 1:100 or thereverse ratio thereof.

In some embodiments, the preparation of glycan therapeutics (e.g.oligosaccharides and polysaccharides) comprises a desired mixture ofthree different monosaccharide glycan units, such as a mixture of, e.g.for glucose-containing glycan-therapeutic preparations, glucose,galactose and arabinose; glucose, galactose and mannose; glucose,galactose and fructose; glucose, galactose and xylose; glucose,galactose and fucose, glucose, galactose and rhamnose; glucose,arabinose, and mannose; glucose, arabinose and fructose; glucose,arabinose and xylose; glucose, arabinose and fucose; glucose, arabinoseand rhamnose; glucose, mannose and fructose; glucose, mannose andxylose; glucose, mannose and fucose; glucose, mannose rhamnose; glucose,fructose and xylose; glucose, fructose and fucose; glucose, fructose andrhamnose; glucose, fucose and rhamnose, e.g. a in a ratio of 1:1:1,1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:6:1, 1:7:1, 1:8:1, 1:9:1, 1:10:1, 1:12:1,1:14:1, 1:16:1, 1:18:1, 1:20:1, 1:1:2, 1:2:2, 1:3:2, 1:4:2, 1:5:2,1:6:2, 1:7:2, 1:8:2, 1:9:2, 1:10:2, 1:1:3, 1:2:3, 1:3:3, 1:4:3, 1:5:3,1:6:3, 1:7:3, 1:8:3, 1:9:3, 1:10:3, 1:1:4, 1:2:4, 1:3:4, 1:4:4, 1:5:4,1:6:4, 1:7:4, 1:8:4, 1:9:4, 1:10:4, 1:1:5, 1:2:5, 1:3:5, 1:4:5, 1:5:5,1:6:5, 1:7:5, 1:8:5, 1:9:5, 1:10:5, etc.

In some embodiments, the preparation of glycan therapeutics (e.g.oligosaccharides and polysaccharides) comprises substantially all diose(2) monosaccharide units, optionally comprising 1%, 2%, 3%, 4% 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% ofa triose (3) tetrose (4), pentose (5), hexose (6), or heptose (7), orany combination thereof.

In some embodiments, the preparation of glycan therapeutics (e.g.oligosaccharides and polysaccharides) comprises substantially all triose(3) monosaccharide units, optionally comprising 1%, 2%, 3%, 4% 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% ofa diose (2), tetrose (4), pentose (5), hexose (6), or heptose (7), orany combination thereof.

In some embodiments, the preparation of glycan therapeutics (e.g.oligosaccharides and polysaccharides) comprises substantially all oftetrose (4) monosaccharide units, optionally comprising 1%, 2%, 3%, 4%5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or20% of a diose (2), triose (3), pentose (5), hexose (6), or heptose (7),or any combination thereof.

In some embodiments, the preparation of glycan therapeutics (e.g.oligosaccharides and polysaccharides) comprises substantially all ofpentose (5) monosaccharide units, optionally comprising 1%, 2%, 3%, 4%5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or20% of a diose (2), triose (3) tetrose (4), hexose (6), or heptose (7),or any combination thereof.

In some embodiments, the preparation of glycan therapeutics (e.g.oligosaccharides and polysaccharides) comprises substantially all ofhexose (6) monosaccharide units, optionally comprising 1%, 2%, 3%, 4%5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or20% of a diose (2), triose (3) tetrose (4), pentose (5), or heptose (7),or any combination thereof.

In some embodiments, the preparation of glycan therapeutics (e.g.oligosaccharides and polysaccharides) comprises substantially all ofheptose (7) monosaccharide units, optionally comprising 1%, 2%, 3%, 4%5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or20% of a diose (2), triose (3) tetrose (4), pentose (5), or hexose (6),or any combination thereof.

In some embodiments, preparations of glycan therapeutics are provided,wherein at least one glycan unit is a furanose sugar. In someembodiments, preparations of glycans are provided, wherein at least oneglycan unit is a pyranose sugar. In some embodiments, glycantherapeutics comprise mixtures of furanose and pyranose sugars. In someembodiments, the furanose: pyranose sugar ratio in a preparation isabout 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1,1:1, 1.2:1, 1.5:1, 1.7:1, 2:1, 2.2:1, 2.5:1, 2.7:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, or about 10:1.

In some embodiments, the preparation of glycan therapeutics (e.g.oligosaccharides and polysaccharides) comprises a desired mixture offuranose and pyranose sugars, e.g. of a desired ratio, such as: 1:1,1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18,1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75,1:80, 1:85, 1:90, 1:100, 1:150 furanose to and pyranose or pyranose tofuranose.

In some embodiments, the preparation of glycan therapeutics comprisessubstantially all furanose or pyranose sugar, optionally comprising 1%,2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, or 20% of the respective other sugar.

In some embodiments, the preparation of glycan therapeutics comprisessubstantially all pyranose sugar and no more than about 0.1%, 02%, 0.5%,1%, 2%, 3%, 4%, or no more than 5% of monomeric glycan units in thepreparation in furanose form. In some embodiments, no more than 3%, 2%or no more than 1% of monomeric glycan units in the preparation are infuranose form.

In some embodiments, the preparation of glycan therapeutics does notcomprise N-acetylgalactosamine or N-acetylglucosamine. In someembodiments, the preparation of glycans does not comprise sialic acid.In some embodiments, the preparation of glycan therapeutics does notcomprise a lipid and fatty acid. In some embodiments, the preparation ofglycan therapeutics does not comprise an amino acid.

In some embodiments, the preparation of glycan therapeutics does notcomprise a detectable repeating unit. In some embodiments, thepreparation of glycan therapeutics does not comprise a statisticallysignificant amount of a repeating unit. In some embodiments, therepeating unit has a DP of at least 2, 3, 4, 5, or at least 6 glycanunits. For example, hyaluronan is a glycosaminoglycan with a repeatingdisaccharide unit consisting of two glucose derivatives, glucuronate(glucuronic acid) and N-acetylglucosamine. The glycosidic linkages arebeta (1→3) and beta (1→4). Cellulose is a polymer made with repeatedglucose units linked together by beta-linkages. The presence and amountof repeating units can be determined, e.g. using by total hydrolysis(e.g. to determine the proportion of glycan units), methylation analysis(e.g. to determine the distribution of bond types), and HSQC (e.g. todetermine the distribution of alpha- and beta-glycosides). Statisticalmethods to determine significance are known by one of skill in the art.

If desired, the monosaccharide or oligosaccharide glycan units of theglycans are further substituted or derivatized, e.g., hydroxyl groupscan be etherified or esterified. For example, the glycans (e.g. oligo-or polysaccharide) can contain modified saccharide units, such as2′-deoxyribose wherein a hydroxyl group is removed, 2′-fluororibosewherein a hydroxyl group is replace with a fluorine, orN-acetylglucosamine, a nitrogen-containing form of glucose (e.g.,2′-fluororibose, deoxyribose, and hexose). The degree of substitution(DS, average number of hydroxyl groups per glycosyl unit) can be 1, 2,or 3, or another suitable DS. In some embodiments, 1%, 2%, 3%, 4% 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% of glycan units are substituted orderivatized. In some embodiments, the degree of substitution variesbetween subunits, e.g., a certain percentage is not derivatized,exhibits a DS of 1, exhibits a DS of 2, or exhibits a DS of 3. Anydesired mixture can be generated, e.g. 0-99% of subunits are notderivatized, 0-99% of subunits exhibit a DS of 1, 0-99% of subunitsexhibit a DS of 2, and 0-99% of subunits exhibit a DS of 3, with thetotal making up 100%. The degree of substitution can be controlled byadjusting the average number of moles of substituent added to a glycosylmoiety (molar substitution (MS)). The distribution of substituents alongthe length of the glycan oligo- or polysaccharide chain can becontrolled by adjusting the reaction conditions, reagent type, andextent of substitution. In some embodiments, the monomeric subunits aresubstituted with one or more of an acetate ester, sulfate half-ester,phosphate ester, or a pyruvyl cyclic acetal group.

The molar percentage of species with a degree of polymerization (DP) ofn (denoted here as DP(n)) in a population is determined by highperformance liquid chromatography (HPLC), e.g., on an Agilent 1260BioInert series instrument equipped with a refractive index (RI)detector and a variety of columns familiar to those skilled in the artusing water as the mobile phase. The columns are selected fromchemistries including, but not limited to, HILIC, metal coordination,and aqueous size-exclusion chromatography that best isolate the speciesof interest. Molar % DP(n) is determined by the formula:% DP(n)=100*AUC[DP(n)]/AUC[DP(total)],

where AUC is defined as the area under the curve for the species ofinterest as determined by calibration to known standards. The molarpercentage of glycosidic bond isomers (% alpha and % beta) aredetermined by nuclear magnetic resonance (NMR) spectroscopy using avariety of 2D techniques familiar to those skilled in the art. Alpha-and beta-isomers may be distinguished, e.g., by their distinct shift onthe NMR spectrum and the molar percentage is determined by the formula:(glycosidic isomer n) of glycosidic bonds=100*AUC[shift (isomern)]/AUC[shift (isomer alpha+isomer beta)],

where AUC is defined as the area under the curve at a specific shiftvalue known to represent the desired isomer n. The molar percentage ofregiochemical isomers is determined in an analogous fashion using theformula:% (regioisomer n) of regioisomers=100*AUC[shift (regioisomern)]/AUC[shift (all regioisomers)].

The relative percentage of monomeric sugars making up the oligomericpopulation is determined, e.g., by total acidic digestion of theoligomeric sample followed by conversion to the alditol acetate followedby gas chromatographic (GC) analysis of the resultant monomericsolutions compared against GC of known standards. The molar percentageof monomer(n), where n can be any sugar, is determined by the formula:% (sugar n)=100*AUC[sugar n]/AUC[total of all monomeric sugars].

In some embodiments, the solubility of the preparation of glycantherapeutics can be controlled, e.g. by selecting the charge, structure(e.g. DP, degree of branching), and/or derivatization of the glycanunits.

Preparations of glycan therapeutics consisting of one type of sugar unituniformly linked in linear chains are usually water insoluble at 23° C.even when the glycans have a low molecular weight with degrees ofpolymerization (DP) between 20 and 30. The degree of solubility of theglycan therapeutics can be adjusted, e.g. by the introduction of(1→6)-linkages and alternating glycosidic bonds in the glycans. Theextra degrees of freedom provided by the rotation about the C-5 to C-6bonds gives higher solution entropy values. Homoglycans with two typesof sugar linkages or heteroglycans composed of two types of sugars aregenerally more soluble than homogeneous polymers. Ionization of linearhomoglycans can add solubility, e.g. to that of gels. The viscosity ofthe solutions often depends on the tertiary structures of the glycans.

In some embodiments, the glycan therapeutic preparations are highlybranched, e.g. have an average DB of at least 0.01, 0.05, or 0.1. Insome embodiments, the glycan therapeutic preparations have an average DBof 0.1 to 0.2. The glycan therapeutic preparations comprising branchedoligosaccharide are highly soluble. In some embodiments, glycantherapeutic preparations can be concentrated to at least to 55 Brix, 65Brix, 60 Brix, 65 Brix, 70 Brix, 75 Brix, 80 Brix, or at least 85 Brixwithout obvious solidification or crystallization at 23° C. (finalsolubility limit). In some embodiments, glycan therapeutic preparationsare concentrated to at least about 0.5 g/ml, 1 g/ml, 1.5 g/ml, 2 g/ml,2.5 g/ml, 3 g/ml, 3.5 g/ml or at least 4 g/ml without obvioussolidification or crystallization at 23° C. (final solubility limit).

In some embodiments, the glycan therapeutic preparations (e.g.oligosaccharides) are branched, e.g. have an average DB of at least0.01, 0.05, or 0.1 and has a final solubility limit in water of at leastabout 70 Brix, 75 Brix, 80 Brix, or at least about 85 Brix at 23° C. oris at least about 1 g/ml, 2 g/ml or at least about 3 g/ml.

In some embodiments, the preparation of glycan therapeutics has a finalsolubility limit of at least 0.001 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L,0.1 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L, 0.5 g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L,0.9 g/L, 1 g/L, 5 g/L, 10 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L, 100 g/L,200 g/L, 300 g/L, 400 g/L, 500 g/L, 600 g/L, 700 g/L, 800 g/L, 900 g/L,1000 g/L in deionized water, or in a suitable buffer such as, e.g.,phosphate-buffered saline, pH 7.4 or similar physiological pH) and at20° C. In some embodiments, the preparation of glycan therapeutics isgreater than 50%, greater than 60%, greater than 70%, greater than 80%,greater than 90%, greater than 95%, greater than 96%, greater than 97%,greater than 98%, greater than 99%, or greater than 99.5% soluble withno precipitation observed at a concentration of greater than 0.001 g/L,0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L, 0.5g/L, 0.6 g/L, 0.7 g/L, 0.8 g/L, 0.9 g/L, 1 g/L, 5 g/L, 10 g/L, 20 g/L,30 g/L, 40 g/L, 50 g/L, 100 g/L, 200 g/L, 300 g/L, 400 g/L, 500 g/L, 600g/L, 700 g/L, 800 g/L, 900 g/L, 1000 g/L in deionized water, or in asuitable buffer such as, e.g., phosphate-buffered saline, pH 7.4 orsimilar physiological pH) and at 20° C.

In some embodiments, the preparation of glycan therapeutics has adesired degree of sweetness. For example, sucrose (table sugar) is theprototype of a sweet substance. Sucrose in solution has a sweetnessperception rating of 1, and other substances are rated relative to this(e.g., fructose, is rated at 1.7 times the sweetness of sucrose). Insome embodiments, the sweetness of the preparation of glycantherapeutics ranges from 0.1 to 500,000 relative to sucrose. In someembodiments, the relative sweetness is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000,10000, 25000, 50000, 75000, 100000, 150000, 200000, 250000, 300000,350000, 40000, 450000, 500000, or more than 500,000 relative to sucrose(with sucrose scored as one). In some embodiments, the preparation ofglycan therapeutics is mildly sweet, or both sweet and bitter.

In some embodiments, the preparation of glycan therapeutics, e.g. apreparation that is substantially DP2+ or DP3+ (e.g. at least 80%, 90%,or at least 95%, or a fractionated preparation of DP2+ or DP3+), issubstantially imperceptible as sweet and the relative sweetness is about0, 0.0001, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,or about 0.8 relative to sucrose (with sucrose scored as one).

Identification and Characterization of Glycan Therapeutic Preparations

If desired, the glycan therapeutic preparations can be characterized.For example, preparations of glycan therapeutics that have beenidentified in one or more in vitro or in vivo assays to increase growthof health promoting bacteria or that suppress the growth of microbialpathogens may be further characterized by any method known in the artand by the methods described herein. Suitable methods are furtherdescribed in the Examples.

For glycan therapeutic preparations, the monomeric building blocks (e.g.the monosaccharide or glycan unit composition), the anomericconfiguration of side chains, the presence and location of substituentgroups, degree of polymerization/molecular weight and the linkagepattern can be identified by standard methods known in the art, such as,e.g. methylation analysis, reductive cleavage, hydrolysis, GC-MS (gaschromatography-mass spectrometry), MALDI-MS (Matrix-assisted laserdesorption/ionization-mass spectrometry), ESI-MS (Electrosprayionization-mass spectrometry), HPLC (High-Performance Liquidchromatography with ultraviolet or refractive index detection),HPAEC-PAD (High-Performance Anion-Exchange chromatography with PulsedAmperometric Detection), CE (capillary electrophoresis), IR (infrared)/Raman spectroscopy, and NMR (Nuclear magnetic resonance)spectroscopy techniques. For polymers of crystalline consistency, thecrystal structure can be solved using, e.g., solid-state NMR, FT-IR(Fourier transform infrared spectroscopy), and WAXS (wide-angle X-rayscattering). The DP, DP distribution, and polydispersity can bedetermined by, e.g., viscosimetry and SEC (SEC-HPLC, high performancesize-exclusion chromatography). Alien groups, end groups andsubstituents can be identified, e.g., using SEC with labeling, aqueousanalytics, MALDI-MS, FT-IR, and NMR. To identify the monomericcomponents of the glycans methods such as, e.g. acid-catalyzedhydrolysis, HPLC (high performance liquid chromatography) or GLC(gas-liquid chromatography) (after conversion to alditol acetates) maybe used. To determine the linkages present in the glycans, in oneexample, the polysaccharide is methylated with methyl iodide and strongbase in DMSO, hydrolysis is performed, a reduction to partiallymethylated alditols is achieved, an acetylation to methylated alditolacetates is performed, and the analysis is carried out by GLC/MS(gas-liquid chromatography coupled with mass spectrometry). In someembodiments, to determine the polysaccharide sequence a partialdepolymerization is carried out using an acid or enzymes to determinethe structures. Possible structures of the polysaccharide are comparedto those of the hydrolytic oligomers, and it is determined which one ofthe possible structures could produce the oligomers. To identify theanomeric configuration, in one example, the intact polysaccharide or apreparation of oligosaccharides are subjected to enzymatic analysis,e.g. they are contacted with an enzyme that is specific for a particulartype of linkage, e.g., β-galactosidase, or α-glucosidase, etc., and NMRmay be used to analyze the products.

For example, the distribution of (or average) degree of polymerization(DP) of a glycan therapeutic preparation may be measured by injecting asample with a concentration of, e.g., 10-100 mg/mL onto an Agilent 1260BioPure HPLC (or similar) equipped with a 7.8×300 mm BioRad AminexHPX-42A column (or similar) and RI detector as described, e.g., in Gómezet al. (Purification, Characterization, and Prebiotic Properties ofPectic Oligosaccharides from Orange Peel Wastes, J Agric Food Chem,2014, 62:9769). Alternatively, a sample with a concentration may beinjected into a Dionex ICS5000 HPLC (or similar) equipped with a 4×250mm Dionex CarboPac PA1 column (or similar) and PAD detector asdescribed, e.g., in Holck et al., (Feruloylated and nonferuloylatedarabino-oligosaccharides from sugar beet pectin selectively stimulatethe growth of bifidobacterium spp. in human fecal in vitrofermentations, Journal of Agricultural and Food Chemistry, 2011, 59(12),6511-6519). Integration of the resulting spectrum compared against astandard solution of oligomers allows determination of the average DP.

Distribution of molecular weights can be measured, e.g, by MALDI massspectrometry. Oligosaccharide concentration can be measured with aMettler-Toledo sugar refractometer (or similar) with the final valueadjusted against a standardized curve to account for refractivedifferences between monomers and oligomers.

Distribution of glycoside regiochemistry can be characterized, e.g., bya variety of 2D-NMR techniques including COSY, HMBC, HSQC, DEPT, andTOCSY analysis using standard pulse sequences and a Bruker 500 MHzspectrometer. Peaks can be assigned by correlation to the spectra ofnaturally occurring polysaccharides with known regiochemistry.

In some embodiments, the relative peak assignment of a sample isdependent on a number of factors including, but not limited to, theconcentration and purity of the sample, the identity and quality of thesolvent (e.g., the isotopically labeled solvent), and the pulse sequenceutilized. As such, in embodiments, the relative peak assignment of, forexample, a glycan comprising glucose may vary (e.g., by about 0.01 ppm,about 0.02 ppm, or about 0.05 ppm) when the NMR spectrum is obtained insimilar conditions due to said factors. In these instances as usedherein, the terms “corresponding peak” or “corresponding peaks” refer toNMR peaks associated with the same sample but that vary (e.g., by about0.01 ppm, about 0.02 ppm, or about 0.05 ppm) due to factors including,for example, the concentration and purity of the sample, the identityand quality of the isotopically labeled solvent, and the pulse sequenceutilized.

Monomeric compositions of oligomers may be measured, e.g., by thecomplete hydrolysis method in which a known amount of oligomer isdissolved into a strong acid at elevated temperature and allowedsufficient time for total hydrolysis to occur. The concentration ofindividual monomers may then be measured by the HPLC or GC methodsdescribed herein and known in the art to achieve relative abundancemeasurements as in Holck et al. Absolute amounts can be measured byspiking the HPLC sample with a known amount of detector active standardselected to prevent overlap with any of the critical signals.

The degree of branching in any given population may be measured by themethylation analysis method established, e.g, by Hakomori (J. Biochem.(Tokyo), 1964, 55, 205). With these data, identification of potentialrepeat units may be established by combining data from the totalhydrolysis, average DP, and methylation analysis and comparing themagainst the DEPT NMR spectrum. Correlation of the number of anomericcarbon signals to these data indicates if a regular repeat unit isrequired to satisfy the collected data as demonstrated, e.g., inHarding, et al. (Carbohydr. Res. 2005, 340, 1107).

Preparation of glycan therapeutics (e.g. those comprising monosaccharideor disaccharide glycan units such as glucose, galactose, fucose, xylose,arabinose, rhamnose, and mannose) may be identified using one, two,three, or four of the following parameters: a) the presence of 2, 3, 4,5, 6, 7 or more (e.g. at least 4 or 5) diagnostic anomeric NMR peakseach representing a different glycosidic bond type, b) an alpha- tobeta-bond ratio between about 0.8 to 1 and about 5 to 1 (e.g. betweenabout 1:1 and 4:1, commonly favoring the alpha bond type), c) at least 2or at least 3 different glycoside regiochemistries from the list of1,2-; 1,3-; 1,4-; and 1,6-substituted and at least 2 or at least 3different glycoside regiochemistries from list of 1,2,3-; 1,2,4-;1,2,6-; 1,3,4-; 1,3,6-; and 1,4,6-substituted, and d) a DP distributionin which at least 50%, 60%, 70% or at least 80% of the individualspecies have a DP of at least 2, at least 3, between 3 and 30 or between5 and 25. In some embodiments, glycan therapeutics represent a uniquestructural class distinct from naturally occurring oligosaccharides. Insome embodiments, glycan therapeutic preparations have novel averageproperties (e.g., DP, DB, alpha:beta glycosidic bond ratio) that aredistinct from naturally occurring preparations of oligosaccharides.These structural features may be quantitated by the methods describedherein. The glycan therapeutic preparations described herein have atleast one, two, three, four, or at least five of the followingcharacteristics:

-   -   (i) a distribution of molecular weights ranging, e.g. from about        DP3 to about DP30 or from about DP5 to about DP25 that may be        identified by quantitative mass spectrometry measurements,        SEC-HPLC, IAC-HPLC, or IEC-HPLC;    -   (ii) a significant proportion of both alpha and beta bonds, with        bond ratios, e.g., ranging from 0.8:1, 1:1, 2:1, 3:1, 4:1, to        5:1 (generally favoring the alpha stereochemistry) that may be        identified by a variety of NMR techniques including the HSQC        pulse sequence which allows explicit discrimination and        quantitation of signals from alpha and beta glycosides. The        presence of both alpha- and beta-glycosidic bonds in the        observed ratios (see Table 6, showing the presence of a large        proportion of both alpha and beta bonds across the single and        multi-sugar glycans tested) in glycan therapeutic preparation of        some embodiments, is distinct from preparations of naturally        occurring oligo- or polysaccharides which generally favor one        primary glycosidic stereochemistry and optionally comprise only        a relatively small portion of the opposing stereochemistry;    -   (iii) presence of at least one, two, three or four glycoside        regiochemistries that may be identified either by a fingerprint        NMR process or by the permethylation branching identification        developed by Hakomori, et al. In some embodiments, glycan        therapeutic preparations have at least 0.1%, 0.2%, 0.5%, 1%, 2%,        3%, 4%, 5%, 6%, 7%, 8%, 9%, or at least 10% of one, two, three        or four of the 1,2-; 1,3-; 1,4-, and 1,6-glycoside bond types.        In some embodiments, glycan therapeutic preparations have at        least 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or        at least 10% of two of the 1,2-; 1,3-; 1,4-, and 1,6-glycoside        bond types. In some embodiments, glycan therapeutic preparations        have at least 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,        9%, or at least 10% of three of the 1,2-; 1,3-; 1,4-, and        1,6-glycoside bond types. In some embodiments, glycan        therapeutic preparations have at least 0.1%, 0.2%, 0.5%, 1%, 2%,        3%, 4%, 5%, 6%, 7%, 8%, 9%, or at least 10% of all four of the        1,2-; 1,3-; 1,4-, and 1,6-glycoside bond types. In some        embodiments, the glycan therapeutic preparation additionally        comprises at least 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4% or at least        5% of branched bond types. In some embodiments, the glycan        therapeutic preparation comprises at least 0.1%, 0.2%, 0.5%, 1%,        2%, 3%, 4% or at least 5% of at least one, two, or at least        three branched bond types including but not limited to 1,3,6-;        1,4,6-; or 1,2,4-glycosides. In some embodiments, the glycan        therapeutic preparation comprises at least two branched bond        types of 1,3,6-; 1,4,6-; or 1,2,4-glycosides. In some        embodiments, the glycan therapeutic preparation comprises at        least 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4% or at least 5% of three        branched bond types of 1,3,6-; 1,4,6-; or 1,2,4-glycosides.        Sugars that do not have a hydroxyl group at a given position X        will not will not have the 1,X-bond type, e.g. fucose        (6-dehydroxy-galactose) will not have 1,6-glycosidic bonds but        will have 1,2-; 1,3-; and 1,4-glycosidic bonds. In some        embodiments, the glycan therapeutic preparation comprises at        least 0.1%, 02%, 0.5%, 1%, 2%, or at least 3% of monomeric        glycan units in furanose form. The presence of a large number of        glycoside regiochemistries and branching (see FIG. 4A, FIG. 4B,        and FIG. 4C for 3 exemplary glycans) in glycan therapeutic        preparation of some embodiments, is distinct from preparations        of naturally occurring oligo- or polysaccharides which generally        favor specific bond architectures. Although all of these        regiochemistries are known to occur in oligosaccharides of        natural sources, preparations of naturally sourced        oligosaccharide do not comprise the number and complexity of        regiochemistries that are exhibited by glycan therapeutic        preparations of some embodiments.    -   (iv) a distribution of glycosidic bonds that represents at least        50%, 60%, 70%, 80% or at least 90% of all possible combinations        of regio- and stereochemistries. Individually, the regiochemical        distribution can be determined by branching analysis and the        stereochemical distribution can be determined by NMR. The        HSQC-NMR. In some embodiments, the glycan therapeutic        preparations exhibit a diversity of peaks in the anomeric region        that are associated with a multiplicative combination of both        regiochemistry and stereochemistry. In some embodiments, the        glycan therapeutic preparation comprises at least two or at        least three of alpha-1,2-; alpha-1,3-; alpha-1,4-; and        alpha-1,6-glycosides and at least two, or at least three of        beta-1,2-; beta-1,3-; beta-1,4-; and beta-1,6-glycosides. In        some embodiments, the glycan therapeutic preparation comprises        all four of alpha-1,2-; alpha-1,3-; alpha-1,4-; and        alpha-1,6-glycosides and all four of beta-1,2-; beta-1,3-;        beta-1,4-; and beta-1,6-glycosides. As an exemplar, HSQC of a        glu100 preparation shows that the preparation contains all        alpha-1,2-; alpha-1,3-; alpha-1,4-; and alpha-1,6-glycosides as        well as all beta-1,2-; beta-1,3-; beta-1,4-; and        beta-1,6-glycosides. Sugars that do not have a hydroxyl group at        a given position X will not will not have the 1,X-bond type,        e.g. fucose (6-dehydroxy-galactose) will not have 1,6-glycosidic        bonds but will have 1,2-; 1,3-; and 1,4-glycosidic bonds;    -   (v) a unique HSQC “fingerprint” that is the result of the        additive nature of the HSQC pulse sequence. For any given        glycan, the HSQC spectra allow the identification of peaks that        are unique to specific regio- and stereochemical bond        arrangement. For example, FIG. 5 shows a partial assignment of        the spectra of a glu100 preparation demonstrating how these        peaks may be used to identify specific glycosidic regio- and        stereochemistries. Component glycan units (e.g. sugars) within a        glycan demonstrate spin-isolation in the HSQC pulse sequence and        the HSQC spectrum of any glycan consisting of multiple sugars is        the sum of peaks of its individual sugars. Glycan unit        constituents (e.g. monomers) can be identified by an HSQC        spectrum that shows 4, 5, 6 or more of the peaks listed in Table        7 for each of its component glycan units (e.g. sugars). The        spectra in FIGS. 3A-3C exemplify this by comparing the spectra        of preparations of glu100, gal100, and glu50gal50.

Pharmaceutical Compositions, Medical Foods, and Unit Dosage Forms

Provided herein are also methods of producing pharmaceuticalcompositions comprising a glycan therapeutic preparation that meets oneor more, two or more, three or more or four or more of thecharacteristics of the preparations described herein (including criteria(i)-(v) above). In particular, methods include providing a glycantherapeutic preparation and acquiring the value(s) for one or more, twoor more, or three or more characteristics of the preparation, including,e.g., i) the degree of polymerization (DP), ii) the average degree ofbranching (DB, branching points per residue), iii) the ratio ofalpha-glycosidic to beta-glycosidic bonds, iv) the identity of theglycan units, and v) the ratio of glycan units, and producing apharmaceutical composition comprising a glycan therapeutic preparationif the desired or predetermined criteria of the preparation are metwithin a desired range of deviation.

Methods for formulating the glycan therapeutic preparation into apharmaceutical composition, medical food or dietary supplement are knownin the art and may include one or more, two or more, three or more, orfour or more of the following steps: (i) formulating the preparationinto drug product, (ii) packaging the preparation, (iii) labeling thepackaged preparation, and (iv) selling or offering for sale the packagedand labeled preparation. Formulating the glycan therapeutic preparationinto a drug product is known in the art and may include one or more, twoor more, three or more, or four or more of the following steps: (i)removing unwanted constituents from the preparation, (ii) reducing thevolume of the preparation, (iii) sterilizing the preparation, (iv)admixing the preparation with a pharmaceutically acceptable excipient orcarrier, (v) admixing the preparation with a second drug orpharmaceutical agent, (vi) formulating the preparation into a suitableconsistency, such as, e.g., aqueous diluted solution, a syrup or asolid, (vii) formulating the preparation into a suitable dosage form,e.g. into a tablet, pill or capsule.

In some embodiments, the glycan therapeutic preparation undergoesfurther processing to produce either glycan therapeutic syrup or powder.For example, in one variation, the glycan therapeutic preparation isconcentrated to form a syrup. Any suitable methods known in the art toconcentrate a solution may be used, such as the use of a vacuumevaporator. In another variation, the glycan therapeutic preparation isspray dried to form a powder. Any suitable methods known in the art tospray dry a solution to form a powder may be used.

Provided herein are pharmaceutical compositions, medical foods anddietary supplements comprising glycan therapeutic preparations.Optionally, the pharmaceutical compositions, medical foods and dietarysupplements comprising glycan therapeutic preparations further comprisea second agent, e.g., a prebiotic substance and/or a probioticbacterium. In some embodiments, the pharmaceutical compositions andmedical foods and dietary supplements comprising glycan therapeuticpreparations further comprise a micronutrient. In some embodiments, thepharmaceutical compositions and medical foods and dietary supplementscomprising glycan therapeutic preparations do not contain a prebioticsubstance. In some embodiments, the pharmaceutical compositions andmedical foods and dietary supplements comprising glycan therapeuticpreparations do not contain a probiotic bacterium. Further, optionally,the pharmaceutical compositions and medical foods and dietarysupplements comprising glycan therapeutic preparations comprise one ormore excipients or carriers, including diluents, binders, disintegrants,dispersants, lubricants, glidants, stabilizers, surfactants, flavoringagents, and colorants.

In some embodiments, the pharmaceutical compositions or medical foodsand dietary supplements comprise a glycan therapeutic preparation ofglu100, ara100, xyl100, gal100, glu50gal50, gal75xyl25, ara50gal50,man62glu38, ara50xyl50, man52glu29gal19, or glu33gal33fuc33.

In some embodiments, the pharmaceutical compositions or medical foodsand dietary supplements comprise a glycan therapeutic preparation ofglu100, ara100, xyl100, glu50gal50, man52glu29gal19, or glu33gal33fuc33.

In some embodiments, the pharmaceutical compositions or medical foodsand dietary supplements comprise a glycan therapeutic preparation ofglu100 and man52glu29gal19.

In some embodiments, pharmaceutical compositions and medical foods anddietary supplements comprising glycan therapeutic preparations (and kitscomprising same) comprise one or more micronutrient. In someembodiments, the micronutrient is selected from the group consisting ofa trace mineral, choline, a vitamin, and a polyphenol.

In some embodiments, the micronutrient is a trace metal. Trace mineralssuitable as a micronutrient include, but are not limited to, boron,cobalt, chromium, calcium, copper, fluoride, iodine, iron, magnesium,manganese, molybdenum, selenium, and zinc.

In some embodiments, the micronutrient is a vitamin. Vitamins suitableas a micronutrient include, but are not limited to, Vitamin B complex,Vitamin B1 (thiamin), Vitamin B2 (riboflavin), Vitamin B3 (niacin),Vitamin B5 (pantothenic acid), Vitamin B6 group (pyridoxine, pyridoxal,pyridoxamine), Vitamin B7 (biotin), Vitamin B8 (ergadenylic acid),Vitamin B9 (folic acid), Vitamin B12 (cyanocobalamin), Choline, VitaminA (retinol), Vitamin C (ascorbic acid), Vitamin D, Vitamin E(tocopherol), Vitamin K, carotenoids (alpha carotene, beta carotene,cryptoxanthin, lutein, lycopene) and zeaxanthin.

In some embodiments, the micronutrient is a polyphenol. Polyphenols arechemical compounds or molecules that are characterized by having atleast one aromatic ring with one or more hydroxyl groups. In someembodiments, the polyphenol is a synthetic polyphenol or a naturallyoccurring polyphenol. In some embodiments, the polyphenol is a naturallyoccurring polyphenol and is derived from plant source material.

In some embodiments, the polyphenol is a flavonoid or catechin. In someembodiments, the flavonoid or catechin is selected from anthocyanins,chalcones, dihydrochalcones, dihydroflavonols, flavanols, flavanones,flavones, flavonols and isoflavonoids. In some embodiments, thepolyphenol is a lignan.

In some embodiments, the polyphenol is selected fromalkylmethoxyphenols, alkylphenols, curcuminoids, furanocoumarins,hydroxybenzaldehydes, hydroxybenzoketones, hydroxycinnamaldehydes,hydroxycoumarins, hydroxyphenylpropenes, methoxyphenols, naphtoquinones,phenolic terpenes, and tyrosols. In some embodiments, the polyphenol isa tannin or tannic acid.

In some embodiments, the polyphenol is selected from hydroxybenzoicacids, hydroxycinnamic acids, hydroxyphenylacetic acids,hydroxyphenylpropanoic acids, and hydroxyphenylpentanoic acids. In someembodiments, the polyphenol is a stilbene.

In some embodiments, the polyphenol is any one of the polyphenols listedin Table 5.

Further, if desired, the pharmaceutical compositions and medical foodsand dietary supplements comprising glycan therapeutic preparations maycomprise therapeutically active agents, prebiotic substances and/orprobiotic bacteria. Alternatively or in addition, therapeutically activeagents, prebiotic substances and/or probiotic bacteria may beadministered separately (e.g. prior to, concurrent with or afteradministration of the glycan therapeutics) and not as a part of thepharmaceutical composition or medical food or dietary supplement (e.g.as a co-formulation) of glycan therapeutics. In some embodiments,pharmaceutical compositions or medical foods or dietary supplementscomprising preparations of glycan therapeutics are administered incombination with a recommended or prescribed diet, e.g. a diet that isrich in probiotic and/or prebiotic-containing foods, such as it may bedetermined by a physician or other healthcare professional.Therapeutically active agents, prebiotic substances and/or probioticbacteria may be administered to modulate the gut microbiome of thesubject. In some embodiments, the combined effect (e.g. on the number orintensity of the microbial, genomic or functional shifts) is additive.In other embodiments, the combined effect (e.g. on the number orintensity of the microbial, genomic or functional shifts) issynergistic.

In some embodiments, the pharmaceutical compositions and medical foodsand dietary supplements comprising glycan therapeutic preparationsdescribed herein further comprise a prebiotic substance or preparationthereof.

In some embodiments, prebiotics may be administered to a subjectreceiving the pharmaceutical compositions or medical foods or dietarysupplements comprising glycan therapeutic preparations described herein.Prebiotics are non-digestible substances that when consumed may providea beneficial physiological effect on the host by selectively stimulatingthe favorable growth or activity of a limited number of indigenousbacteria in the gut (Gibson G R, Roberfroid M B. Dietary modulation ofthe human colonic microbiota: introducing the concept of prebiotics. JNutr. 1995 June; 125(6):1401-12.). A prebiotic such as a dietary fiberor prebiotic oligosaccharide (e.g. crystalline cellulose, wheat bran,oat bran, corn fiber, soy fiber, beet fiber and the like) may furtherencourage the growth of probiotic and/or commensal bacteria in the gutby providing a fermentable dose of carbohydrates to the bacteria andincrease the levels of those microbial populations (e.g. lactobacilliand bifidobacteria) in the gastrointestinal tract.

Prebiotics include, but are not limited to, various galactans andcarbohydrate based gums, such as psyllium, guar, carrageen, gellan,lactulose, and konjac. In some embodiments, the prebiotic is one or moreof galactooligosaccharides (GOS), lactulose, raffinose, stachyose,lactosucrose, fructo-oligosaccharides (FOS, e.g. oligofructose oroligofructan), inulin, isomalto-oligosaccharide, xylo-oligosaccharides(XOS), paratinose oligosaccharide, isomaltose oligosaccharides (IMOS),transgalactosylated oligosaccharides (e.g.transgalacto-oligosaccharides), transgalactosylate disaccharides,soybean oligosaccharides (e.g. soyoligosaccharides), chitosanoligosaccharide (chioses), gentiooligosaccharides, soy- andpectin-oligosaccharides, glucooligosaccharides, pecticoligosaccharides,palatinose polycondensates, difructose anhydride III, sorbitol,maltitol, lactitol, polyols, polydextrose, linear and branched dextrans,pullalan, hemicelluloses, reduced paratinose, cellulose, beta-glucose,beta-galactose, beta-fructose, verbascose, galactinol, xylan, inulin,chitosan, beta-glucan, guar gum, gum arabic, pectin, high sodiumalginate, and lambda carrageenan, or mixtures thereof.

Prebiotics can be found in certain foods, e.g. chicory root, Jerusalemartichoke, Dandelion greens, garlic, leek, onion, asparagus, wheat bran,wheat flour, banana, milk, yogurt, sorghum, burdock, broccoli, Brusselssprouts, cabbage, cauliflower, collard greens, kale, radish andrutabaga, and miso. In some embodiments, the glycan therapeuticsdescribed herein are administered to a subject in conjunction with adiet that includes foods rich in prebiotics. Suitable sources of solubleand insoluble fibers are commercially available.

In some embodiments, the pharmaceutical compositions and medical foodsand dietary supplements comprising glycan therapeutic preparationsfurther comprise a probiotic bacterium or preparation thereof, e.g.,derived from bacterial cultures that are generally recognized as safe(GRAS) or known commensal or probiotic microbes. In some embodiments, tomaximize the beneficial effect of endogenous commensal microbes orexogenously administered probiotic microorganisms, the pharmaceuticalcompositions and medical foods and dietary supplements comprising glycantherapeutic preparations are administered to stimulate the growth and/oractivity of advantageous bacteria in the GI tract.

Examples of suitable probiotics include, but are not limited to,organisms classified as genera Bacteroides, Blautia, Clostridium,Fusobacterium, Eubacterium, Ruminococcus, Peptococcus,Peptostreptococcus, Akkermansia, Faecalibacterium, Roseburia,Prevotella, Bifidobacterium, Lactobacillus, Bacillus, Enterococcus,Escherichia, Streptococcus, Saccharomyces, Streptomyces, and familyChristensenellaceae. Non-exclusive examples of probiotic bacteria thatcan be used in the methods and compositions described herein include L.acidophilus, Lactobacillus species, such as L. crispatus, L. casei, L.rhamnosus, L. reuteri, L. fermentum, L. plantarum, L. sporogenes, and L.bulgaricus, as well as Bifidobacterum species, such as B. lactis, B.animalis, B. bifidum, B. longum, B. adolescentis, and B. infantis.Yeasts, such as Saccharomyces boulardii, are also suitable as probioticsfor administration to the gut, e.g. via oral dosage forms or foods. Forexample, yogurt is a product which already contains bacteria species,such as Lactobacillus bulgaricus and Streptococcus thermophilus.

Beneficial bacteria for the modulation of the gastrointestinalmicrobiota may include bacteria that produce organic acids (lactic &acetic acids) or that produce cytotoxic or cytostatic agents (to inhibitpathogenic growth), such as, e.g., hydrogen peroxide (H₂O₂) andbacteriocins. Bacteriocins are small antimicrobial peptides which cankill both closely-related bacteria, or exhibit a broader spectrum ofactivity (e.g., nisin).

Beneficial bacteria may include one or more of the genus Akkermansia,Anaerofilum, Bacteroides, Blautia, Bifidobacterium, Butyrivibrio,Clostridium, Coprococcus, Dialister, Dorea, Fusobacterium, Eubacterium,Faecalibacterium, Lachnospira, Lactobacillus, Phascolarctobacterium,Peptococcus, Peptostreptococcus, Prevotella, Roseburia, Ruminococcus,and Streptococcus, and/or one or more of the species Akkermansiamuniciphilia, minuta, Clostridium coccoides, Clostridium leptum,Clostridium scindens, Dialister invisus, Eubacterium rectal, Eubacteriumeligens, Faecalibacterium prausnitzii, Streptococcus salivarius, andStreptococcus thermophilus. In some embodiments, the probiotic orcommensal bacteria include one or more of the bacteria listed in Table1.

The prebiotic substances and probiotic strains that may be combined withglycan therapeutics described herein to produce a composition or kit maybe isolated at any level of purity by standard methods and purificationcan be achieved by conventional means known to those skilled in the art,such as distillation, recrystallization and chromatography. Thecultivated bacteria to be used in the composition are separated from theculture broth with any method including, without limitations,centrifuging, filtration or decantation. The cells separated from thefermentation broth are optionally washed by water, saline (0.9% NaCl) orwith any suitable buffer. The wet cell mass obtained may be dried by anysuitable method, e.g., by lyophilization.

In some embodiments, the probiotic bacteria are lyophilized vegetativecells. In some embodiments, the preparations of spores from sporulatingprobiotic bacteria are used.

In one embodiment, a pharmaceutical glycan therapeutic compositionfurther comprises a prebiotic and probiotic. In one embodiment, thepharmaceutical composition comprises probiotics whose viability has beenpartially attenuated (e.g. a mixture comprising 10%, 20%, 30%, 40%, 50%or more non-viable bacteria), or probiotics consisting solely ofnon-viable microbes. The compositions may further comprise microbialmembranes and/or cell walls that have been isolated and purified fromkilled microbes. If desired, the probiotic organism can be incorporatedinto the pharmaceutical glycan therapeutic composition as a culture inwater or another liquid or semisolid medium in which the probioticremains viable. In another technique, a freeze-dried powder containingthe probiotic organism may be incorporated into a particulate materialor liquid or semisolid material by mixing or blending.

In some embodiments, the pharmaceutical compositions and medical foodsand dietary supplements comprising glycan therapeutic preparationsfurther comprise a second therapeutic agent or preparation thereof. Insome embodiments, the therapeutic agent is an antibiotic, an antifungalagent, an antiviral agent, or an anti-inflammatory agent (e.g. acytokine, hormone, etc.). Antibiotics include aminoglycosides, such asamikacin, gentamicin, kanamycin, neomycin, streptomycin, and tobramycin;cephalosporins, such as cefamandole, cefazolin, cephalexin,cephaloglycin, cephaloridine, cephalothin, cephapirin, and cephradine;macrolides, such as erythromycin and troleandomycin; penicillins, suchas penicillin G, amoxicillin, ampicillin, carbenicillin, cloxacillin,dicloxacillin, methicillin, nafcillin, oxacillin, phenethicillin, andticarcillin; polypeptide antibiotics, such as bacitracin,colistimethate, colistin, polymyxin B; tetracyclines, such aschlortetracycline, demeclocycline, doxycycline, methacycline,minocycline, tetracycline, and oxytetracycline; and miscellaneousantibiotics such as chloramphenicol, clindamycin, cycloserine,lincomycin, rifampin, spectinomycin, vancomycin, viomycin andmetronidazole.

The glycan therapeutic preparations described herein, othertherapeutically active agents, prebiotic substances, micronutrients andprobiotics may be comingled or mixed in a single pharmaceuticalcomposition or medical food or dietary supplement. In other embodiments,they may be contained in separate containers (and/or in various suitableunit dosage forms) but packaged together in one or more kits. In someembodiments, the preparations or compositions are not packaged or placedtogether. A physician may then administer the preparations orcompositions together, e.g. prior to, concomitant with, or after oneanother. In some embodiments, the preparations or compositions actsynergistically in modulating the microbiota in a subject, e.g., in theGI tract.

In some embodiments, a pharmaceutical composition comprises between 0.1%and 100% glycan therapeutic preparation by w/w, w/v, v/v or molar %. Inanother embodiment, a pharmaceutical composition comprises about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% ofglycan therapeutic preparation by w/w, w/v, v/v or molar %. In oneembodiment, a pharmaceutical composition comprises about 1-90%, about10-90%, about 20-90%, about 30-90%, about 40-90%, about 40-80%, about40-70%, about 40-60%, about 40-50%, about 50-90%, about 50-80%, about50-70%, about 50-60%, about 60-90%, about 60-80%, about 60-70%, about70-90%, about 70-80%, about 70-90%, about 70-80%, about 80-90%, about90-96%, about 93-96%, about 93-95%, about 94-98%, about 93-99%, or about90-100% of glycan therapeutic preparation by w/w, w/v, v/v or molar %.

A pharmaceutical composition comprising a glycan therapeutic preparationcan optionally comprise one or more excipients or carriers. Thepharmaceutical composition can comprise from about 1% to about 90% ofthe one or more excipients or carriers by w/w, w/v, v/v or molar %. Forexample, the pharmaceutical composition can comprise about 1-90%, 1-75%,1-60%, 1-55%, 1-50%, 1-45%, 1-40%, 1-25%, 1-15%, 1-10%, 10-90%, 10-75%,10-60%, 10-55%, 10-50%, 10-45%, 10-40%, 10-25%, 10-15%, 15-90%, 15-75%,15-60%, 15-55%, 15-50%, 15-45%, 15-40%, 15-25%, 25-90%, 25-75%, 25-60%,25-55%, 25-50%, 25-45%, 25-40%, 40-90%, 40-75%, 40-60%, 40-55%, 40-50%,40-45%, 45-90%, 45-75%, 45-60%, 45-55%, 45-50%, 50-90%, 50-75%, 50-60%,50-55%, 55-90%, 55-75%, 55-60%, 60-90%, 60-75%, 75-90% of the one ormore excipients or carriers by w/w, w/v, v/v or molar %.

Medical Food

Also provided herein are preparations of glycan therapeutics formulatedas a medical food. Any glycan therapeutic preparation described hereinmay be formulated as a medical food as well as pharmaceuticalcompositions that comprise glycan therapeutic preparations.

A medical food is defined in section 5(b)(3) of the Orphan Drug Act (21U.S.C. 360ee(b)(3)). Medical food is formulated to be consumed (oralintake) or administered enterally (e.g. feeding/nasogastric tube) undermedical supervision, e.g. by a physician. It is intended for thespecific dietary management of a disease or condition, such as, e.g.dysbiosis of the gastrointestinal microbiota or a GI-tract diseasedescribed herein. Medical foods as used herein do not include food thatis merely recommended by a physician as part of an overall diet tomanage the symptoms or reduce the risk of a disease or condition.Medical foods comprising a preparation of glycan therapeutics are foodsthat are synthetic (e.g., formulated and/or processed products, such as,being formulated for the partial or exclusive feeding of a patient byoral intake or enteral feeding by tube) and not naturally occurringfoodstuff used in a natural state. Medical foods comprising apreparation of glycan therapeutics may represent a major component ofthe management of a GI tract disease or condition, e.g. the medical foodmay represent a partial or exclusive source of food for the subject inneed of a medical food. In some embodiments, the subject has limited orimpaired capacity to ingest, digest, absorb, or metabolize ordinaryfoodstuffs or certain nutrients. In other embodiments, the subject hasother special medically determined nutrient requirements, the dietarymanagement of which cannot be achieved by the modification of the normaldiet alone. Medical foods comprising a preparation of glycantherapeutics are administered to a subject in need thereof under medicalsupervision (which may be active and ongoing) and usually, the subjectreceives instructions on the use of the medical food. Medical foods maycomprise one or more food additives, color additives, GRAS excipientsand other agents or substances suitable for medical foods. Medical foodpreparations may be nutritionally complete or incomplete formulas.

Dietary Supplements

Any glycan therapeutic preparation described herein may be formulated asa dietary supplement, e.g, for use in a method described herein.

Dietary supplements are regulated under the Dietary Supplement Healthand Education Act (DSHEA) of 1994. A dietary supplement is a producttaken by mouth that contains a “dietary ingredient” intended tosupplement the diet. The “dietary ingredients” in these products mayinclude, in addition to a glycan therapeutic preparation describedherein, one or more of: vitamins, minerals, herbs or other botanicals,amino acids, and substances such as enzymes, organ tissues, glandulars,and metabolites. Dietary supplements can also be extracts orconcentrates, and may be found in many forms such as tablets, capsules,softgels, gelcaps, liquids, or powders. They can also be in other forms,such as a bar, but if they are, information on their label must notrepresent the product as a conventional food or a sole item of a meal ordiet. DSHEA requires that every supplement be labeled a dietarysupplement and not as a general food.

Dosage Forms

The glycan therapeutic preparations described herein may be formulatedinto any suitable dosage form, e.g. for oral or enteral administration.The dosage forms described herein can be manufactured using processesthat are known to those of skill in the art.

The dosage form may be a packet, such as any individual container thatcontains a pharmaceutical glycan therapeutic composition in the form of,e.g., a liquid (wash/rinse), a gel, a cream, an ointment, a powder, atablet, a pill, a capsule, a depository, a single-use applicator ormedical device (e.g. a syringe). For example, provided is also anarticle of manufacture, such as a container comprising a unit dosageform of the pharmaceutical glycan therapeutic composition, and a labelcontaining instructions for use of such glycan therapeutic.

Forms of the compositions that can be used orally include tablets,push-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. Tablets canbe made by compression or molding, optionally with one or more accessoryingredients. Compressed tablets can be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with binders (e.g., povidone,gelatin, hydroxypropylmethyl cellulose), inert diluents, preservative,antioxidant, disintegrant (e.g., sodium starch glycolate, cross-linkedpovidone, cross-linked sodium carboxymethyl cellulose) or lubricating,surface active or dispersing agents. Molded tablets can be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets can optionally becoated or scored and can be formulated so as to provide slow orcontrolled release of the active ingredient therein. Tablets canoptionally be provided with an enteric coating, to provide release inparts of the gut (e.g., colon, lower intestine) other than the stomach.All formulations for oral administration can be in dosages suitable forsuch administration. The push-fit capsules can contain the activeingredients in admixture with filler such as lactose, binders such asstarches, and/or lubricants such as talc or magnesium stearate and,optionally, stabilizers. In soft capsules, the active compounds and/orother agents (e.g., prebiotics or probiotics) can be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers can be added.Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions can be used, which can optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments can be added to thetablets or Dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with watersoluble carrier such as polyethylene glycol or an oil medium, forexample peanut oil, liquid paraffin, or olive oil.

In one embodiment, a provided glycan therapeutic composition includes asoftgel formulation. A softgel can contain a gelatin based shell thatsurrounds a liquid fill. The shell can be made of gelatin, plasticizer(e.g., glycerin and/or sorbitol), modifier, water, color, antioxidant,or flavor. The shell can be made with starch or carrageenan. The outerlayer can be enteric coated. In one embodiment, a softgel formulationcan include a water or oil soluble fill solution, or suspension of acomposition covered by a layer of gelatin.

Solid formulations for oral use may comprise an enteric coating, whichmay control the location at which a glycan therapeutic composition isabsorbed in the digestive system. For example, an enteric coating can bedesigned such that a glycan therapeutic composition does not dissolve inthe stomach but rather travels to the small intestine, where itdissolves. An enteric coating can be stable at low pH (such as in thestomach) and can dissolve at higher pH (for example, in the smallintestine). Material that can be used in enteric coatings includes, forexample, alginic acid, cellulose acetate phthalate, plastics, waxes,shellac, and fatty acids (e.g., stearic acid, palmitic acid).

Formulations for oral use may also be presented in a liquid dosage from.Liquid preparations can be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions syrups or elixirs, or can be presentedas a dry product for reconstitution with water or other suitable vehiclebefore use. Such liquid preparations can contain conventional additives,such as suspending agents, for example sorbitol, methyl cellulose,glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose,aluminum stearate gel or hydrogenated edible fats, emulsifying agents,for example lecithin, sorbitan monooleate, acacia; nonaqueous vehicles(which can include edible oils), for example almond oil, oily esterssuch as glycerine, propylene glycol, or ethyl alcohol; preservatives,for example methyl or propyl p-hydoxybenzoate or sorbic acid, and, ifdesired, conventional flavoring or coloring agents. In some embodiments,liquid formulations can comprise, for example, an agent inwater-in-solution and/or suspension form; and a vehicle comprisingpolyethoxylated castor oil, alcohol, and/or a polyoxyethylated sorbitanmono-oleate with or without flavoring. Each dosage form may comprise aneffective amount of a glycan therapeutic and can optionally comprisepharmaceutically inert agents, such as conventional excipients,vehicles, fillers, binders, disintegrants, pH adjusting substances,buffer, solvents, solubilizing agents, sweeteners, coloring agents, andany other inactive agents that can be included in pharmaceutical dosageforms for administration. Examples of such vehicles and additives can befound in Remington's Pharmaceutical Sciences, 17th edition (1985).

The pharmaceutical compositions provided herein can be in unit-dosageforms or multiple-dosage forms. A unit-dosage form, as used herein,refers to physically discrete unit suitable for administration to humanin need thereof. In an embodiment, the unit-dosage form is provided in apackage. Each unit-dose can contain a predetermined quantity of anactive ingredient(s) sufficient to produce the desired therapeuticeffect, in association with other pharmaceutical carriers or excipients.Examples of unit-dosage forms include, but are not limited to, ampoules,syringes, and individually packaged tablets and capsules. Unit-dosageforms can be administered in fractions or multiples thereof. Amultiple-dosage form is a plurality of identical unit-dosage formspackaged in a single container, which can be administered in segregatedunit-dosage form. Examples of multiple-dosage forms include, but are notlimited to, vials, bottles of tablets or capsules, or bottles of pintsor gallons. In another embodiment the multiple dosage forms comprisedifferent pharmaceutically active agents. For example a multiple dosageform can be provided which comprises a first dosage element comprising acomposition comprising a glycan therapeutic and a second dosage elementcomprising a prebiotic, a therapeutic agent and/or a probiotic, whichcan be in a modified release form. In this example a pair of dosageelements can make a single unit dosage. In one embodiment a kit isprovided comprising multiple unit dosages, wherein each unit comprises afirst dosage element comprising a composition comprising a glycantherapeutic and a second dosage element comprising probiotic, apharmaceutical agent, a prebiotic or a combination thereof, which can bein a modified release form. In another embodiment the kit furthercomprises a set of instructions.

In some embodiments, the unit-dosage form comprises between about 0.001mg to about 10 g of the glycan therapeutic (e.g., a glycan therapeuticdisclosed herein). For example, the unit-dosage form may comprise about0.001 mg to about 9.5 g, about 0.005 mg to about 9 g, about 0.01 mg toabout 8.5 g, about 0.05 mg to about 8 g, about 0.075 mg to about 7.5 g,about 0.1 mg to about 7 g, about 0.25 mg to about 6.5 g, about 0.5 mg toabout 6 g, about 0.75 mg to about 5.5 g, about 1 mg to about 5 g, about2.5 mg to about 4.5 g, about 5 mg to about 4 g, about 7.5 mg to about3.5 g, about 10 mg to about 3 g, about 12.5 mg to about 2.5 g, about 15mg to about 2 g, about 17.5 mg to about 1.5 g, about 20 mg to about 1 g,about 25 mg to about 750 mg, about 50 mg to about 500 g, or about 75 mgto about 250 mg of the glycan therapeutic.

In certain embodiments, the unit-dosage form comprises about 0.001 mg toabout 100 mg, about 0.005 mg to about 75 mg, about 0.01 mg to about 50mg, about 0.05 mg to about 25 mg, about 0.1 mg to about 10 mg, about 0.5mg to about 7.5 mg, or about 1 mg to about 5 mg of the glycantherapeutic. In other embodiments, the unit-dosage form comprises about1 mg to about 100 mg, about 2.5 mg to about 75 mg, about 5 mg to about50 mg, or about 10 mg to about 25 mg of the glycan therapeutic. In otherembodiments, the unit-dosage form comprises about 100 mg to about 10 g,about 250 mg to about 7.5 g, about 500 mg to about 5 g, about 750 mg toabout 2.5 g, or about 1 g to about 2 g of the glycan therapeutic.

In other embodiments, the unit-dosage form comprises between about 0.001mL to about 1000 mL of the glycan therapeutic (e.g., a glycantherapeutic disclosed herein). For example, the unit-dosage form maycomprise about 0.001 mL to about 950 mL, about 0.005 mL to about 900 mL,about 0.01 mL to about 850 mL, about 0.05 mL to about 800 mL, about0.075 mL to about 750 mL, about 0.1 mL to about 700 mL, about 0.25 mL toabout 650 mL, about 0.5 mL to about 600 mL, about 0.75 mL to about 550mL, about 1 mL to about 500 mL, about 2.5 mL to about 450 mL, about 5 mLto about 400 mL, about 7.5 mL to about 350 mL, about 10 mL to about 300mL, about 12.5 mL to about 250 mL, about 15 mL to about 200 mL, about17.5 mL to about 150 mL, about 20 mL to about 100 mL, or about 25 mL toabout 75 mL of the glycan therapeutic.

In certain embodiments, the unit-dosage form comprises about 0.001 mL toabout 10 mL, about 0.005 mL to about 7.5 mL, about 0.01 mL to about 5mL, about 0.05 mL to about 2.5 mL, about 0.1 mL to about 1 mL, about0.25 mL to about 1 mL, or about 0.5 mL to about 1 mL of the glycantherapeutic. In other embodiments, the unit-dosage form comprises about0.01 mL to about 10 mL, about 0.025 mL to about 7.5 mL, about 0.05 mL toabout 5 mL, or about 0.1 mL to about 2.5 mL of the glycan therapeutic.In other embodiments, the unit-dosage form comprises about 0.1 mL toabout 10 mL, about 0.25 mL to about 7.5 mL, about 0.5 mL to about 5 mL,about 0.5 mL to about 2.5 mL, or about 0.5 mL to about 1 mL of theglycan therapeutic.

In some embodiments, the unit-dosage form, e.g., a tablet, capsule(e.g., a hard capsule, push-fit capsule, or soft capsule), or softgel,has a body length of between about 0.1 inches to about 1.5 inches (e.g.,about 0.5 inches and about 1 inch), or about 5 mm to about 50 mm (e.g.,about 10 mm to about 25 mm). In some embodiments, the unit-dosage form.e.g., a tablet, capsule (e.g., a hard capsule, push-fit capsule, or softcapsule), or softgel, has an external diameter of about 0.05 inches toabout 1 inch (e.g., about 0.1 inches to about 0.5 inches), or about 1 mmto about 25 mm (e.g., about 5 mm to about 10 mm).

Each unit-dosage form of the glycan therapeutic may have a caloric valueof between about 0.01 kcal and about 1000 kcal. For example, theunit-dosage form may have a caloric value of about 0.01 kcal to about900 kcal, about 0.05 kcal to about 800 kcal, about 0.1 kcal to about 700kcal, about 0.25 kcal to about 600 kcal, about 0.5 kcal to about 500kcal, about 0.75 kcal to about 400 kcal, about 1 kcal to 300 kcal, about5 kcal to about 200 kcal, or about 10 kcal to about 100 kcal. In certainembodiments, the unit-dosage form of the glycan therapeutic has acaloric value of between 10 kcal to about 500 kcal. In otherembodiments, the unit-dosage form of the glycan therapeutic has acaloric value of between 50 kcal to about 500 kcal.

In still other embodiments, the unit-dosage form may have a caloricvalue of about 0.001 kcal to about 100 kcal, about 0.005 kcal to about90 kcal, about 0.01 kcal to about 80 kcal, about 0.025 kcal to about 70kcal, about 0.05 kcal to about 60 kcal, about 0.075 kcal to about 50kcal, about 0.1 kcal to 40 kcal, about 0.25 kcal to about 30 kcal, about0.5 kcal to about 25 kcal, about 0.25 kcal to about 20 kcal, or about0.1 kcal to about 10 kcal.

The unit-dosage form of the glycan therapeutic may be formulated todissolve in an aqueous solution (e.g., water, milk, juice, and the like)and is orally administered as a beverage, syrup, solution, orsuspension. For example, the unit-form dosage of the glycan therapeuticmay comprise a cube, packet, lozenge, pill, tablet, capsule, candy,powder, elixir, or concentrated syrup formulated for dissolving into anaqueous solution prior to oral administration. In other embodiments, theunit-dosage form of the glycan therapeutic may comprise a cube, packet,lozenge, pill, tablet, capsule, candy, powder, elixir, or concentratedsyrup formulated to dissolve in vivo, e.g., in the mouth, stomach,intestine, or colon of the subject upon oral administration.

In some embodiments, the glycan therapeutic composition is administeredenterically. This preferentially includes oral administration, or by anoral or nasal tube (including nasogastric, nasojejunal, oral gastric, ororal jejunal). In other embodiments, administration includes rectaladministration (including enema, suppository, or colonoscopy).

The dosage forms described herein can be manufactured using processesthat are known to those of skill in the art. For example, for themanufacture of tablets, an effective amount of a prebiotic can bedispersed uniformly in one or more excipients or additives, for example,using high shear granulation, low shear granulation, fluid bedgranulation, or by blending for direct compression. Excipients andadditives include diluents, binders, disintegrants, dispersants,lubricants, glidants, stabilizers, surfactants, antiadherents, sorbents,sweeteners, and colorants, or a combination thereof. Diluents, alsotermed fillers, can be used to increase the bulk of a tablet so that apractical size is provided for compression. Non-limiting examples ofdiluents include lactose, cellulose, microcrystalline cellulose,mannitol, dry starch, hydrolyzed starches, powdered sugar, talc, sodiumchloride, silicon dioxide, titanium oxide, dicalcium phosphatedihydrate, calcium sulfate, calcium carbonate, alumina and kaolin.Binders can impart cohesive qualities to a tablet formulation and can beused to help a tablet remain intact after compression. Non-limitingexamples of suitable binders include starch (including corn starch andpregelatinized starch), gelatin, sugars (e.g., glucose, dextrose,sucrose, lactose and sorbitol), celluloses, polyethylene glycol, alginicacid, dextrin, casein, methyl cellulose, waxes, natural and syntheticgums, e.g., acacia, tragacanth, sodium alginate, gum arabic, xantan gum,and synthetic polymers such as polymethacrylates, polyvinyl alcohols,hydroxypropylcellulose, and polyvinylpyrrolidone. Lubricants can alsofacilitate tablet manufacture; non-limiting examples thereof includemagnesium stearate, calcium stearate, stearic acid, glyceryl behenate,and polyethylene glycol. Disintegrants can facilitate tabletdisintegration after administration, and non-limiting examples thereofinclude starches, alginic acid, crosslinked polymers such as, e.g.,crosslinked polyvinylpyrrolidone, croscarmellose sodium, potassium orsodium starch glycolate, clays, celluloses (e.g.,carboxymethylcelluloses (e.g., carboxymethylcellulose (CMC), CMC-Na,CMC-Ca)), starches, gums and the like. Non-limiting examples of suitableglidants include silicon dioxide, talc, and the like. Stabilizers caninhibit or retard drug decomposition reactions, including oxidativereactions. Surfactants can also include and can be anionic, cationic,amphoteric or nonionic. Exemplary sweeteners may include stevia extract,aspartame, sucrose, alitame, saccharin, and the like. If desired, thetablets can also comprise nontoxic auxiliary substances such as pHbuffering agents, preservatives, e.g., antioxidants, wetting oremulsifying agents, solubilizing agents, coating agents, flavoringagents (e.g., mint, cherry, anise, peach, apricot, licorice, raspberry,vanilla), and the like. Additional excipients and additives may includealuminum acetate, benzyl alcohol, butyl paraben, butylated hydroxytoluene, calcium disodium EDTA, calcium hydrogen phosphate dihydrate,dibasic calcium phosphate, tribasic calcium phosphate, candelilla wax,carnuba wax, castor oil hydrogenated, cetylpyridine chloride, citricacid, colloidal silicone dioxide, copolyvidone, corn starch, cysteineHCl, dimethicone, disodium hydrogen phosphate, erythrosine sodium, ethylcellulose, gelatin, glycerin, glyceryl monooleate, glycerylmonostearate, glycine, HPMC pthalate, hydroxypropylcellulose, hydroxylpropyl methyl cellulose, hypromellose, iron oxide red or ferric oxide,iron oxide yellow, iron oxide or ferric oxide, magnesium carbonate,magnesium oxide, magnesium stearate, methionine, methacrylic acidcopolymer, methyl paraben, silicified microcrystalline cellulose,mineral oil, phosphoric acid, plain calcium phosphate, anhydrous calciumphosphate, polaxamer 407, polaxamer 188, plain polaxamer, polyethyleneoxide, polyoxyl40 stearate, polysorbate 80, potassium bicarbonate,potassium sorbate, potato starch, povidone, propylene glycol, propyleneparaben, propyl paraben, retinyl palmitate, saccharin sodium, selenium,silica, silica gel, fumed silica, sodium benzoate, sodium carbonate,sodium citrate dihydrate, sodium crossmellose, sodium lauryl sulfate,sodium metabisulfite, sodium propionate, sodium starch, sodium starchglycolate, sodium stearyl fumarate, sorbic acid, sorbitol, sorbitanmonooleate, pregelatinized starch, succinic acid, triacetin, triethylcitrate, vegetable stearin, vitamin A, vitamin E, vitamin C, or acombination thereof. The amounts of these excipients and additives canbe properly selected based on their relation to other components andproperties of the preparation and production method.

Immediate-release formulations of an effective amount of a glycantherapeutic composition can comprise one or more combinations ofexcipients that allow for a rapid release of a pharmaceutically activeagent (such as from 1 minute to 1 hour after administration).Controlled-release formulations (also referred to as sustained release(SR), extended-release (ER, XR, or XL), time-release or timed-release,controlled-release (CR), or continuous-release) refer to the release ofa glycan therapeutic composition from a dosage form at a particulardesired point in time after the dosage form is administered to asubject.

In one embodiment a controlled release dosage form begins its releaseand continues that release over an extended period of time. Release canoccur beginning almost immediately or can be sustained. Release can beconstant, can increase or decrease over time, can be pulsed, can becontinuous or intermittent, and the like. In one embodiment, acontrolled release dosage refers to the release of an agent from acomposition or dosage form in which the agent is released according to adesired profile over an extended period of time. In one aspect,controlled-release refers to delayed release of an agent from acomposition or dosage form in which the agent is released according to adesired profile in which the release occurs after a period of time.

Pharmaceutical carriers or vehicles suitable for administration of thecompounds provided herein include all such carriers known to thoseskilled in the art to be suitable for the particular mode ofadministration. In addition, the compositions can one or more componentsthat do not impair the desired action, or with components thatsupplement the desired action, or have another action.

In a further aspect, the dosage form can be an effervescent dosage form.Effervescent means that the dosage form, when mixed with liquid,including water and saliva, evolves a gas. Some effervescent agents (oreffervescent couple) evolve gas by means of a chemical reaction whichtakes place upon exposure of the effervescent disintegration agent towater or to saliva in the mouth. This reaction can be the result of thereaction of a soluble acid source and an alkali monocarbonate orcarbonate source. The reaction of these two general compounds producescarbon dioxide gas upon contact with water or saliva. An effervescentcouple (or the individual acid and base separately) can be coated with asolvent protective or enteric coating to prevent premature reaction.Such a couple can also be mixed with previously lyophilized particles(such as a glycan therapeutic). The acid sources can be any which aresafe for human consumption and can generally include food acids, acidand hydrite antacids such as, for example: citric, tartaric, amalic,fumeric, adipic, and succinics. Carbonate sources include dry solidcarbonate and bicarbonate salt such as sodium bicarbonate, sodiumcarbonate, potassium bicarbonate and potassium carbonate, magnesiumcarbonate and the like. Reactants which evolve oxygen or other gassesand which are safe for human consumption are also included. In oneembodiment citric acid and sodium bicarbonate are used.

In another aspect, the dosage form can be in a candy form (e.g.,matrix), such as a lollipop or lozenge. In one embodiment an effectiveamount of a glycan therapeutic is dispersed within a candy matrix. Inone embodiment the candy matrix comprises one or more sugars (such asdextrose or sucrose). In another embodiment the candy matrix is asugar-free matrix. The choice of a particular candy matrix is subject towide variation. Conventional sweeteners (e.g., sucrose), sugar alcoholssuitable for use with diabetic patients (e.g., sorbitol or mannitol), orother sweeteners (e.g., sweeteners described herein) may be employed.The candy base can be very soft and fast dissolving, or can be hard andslower dissolving. Various forms will have advantages in differentsituations.

A candy mass composition comprising an effective amount of the glycantherapeutic can be orally administered to a subject in need thereof sothat an effective amount of the glycan therapeutic will be released intothe subject's mouth as the candy mass dissolves and is swallowed. Asubject in need thereof includes a human adult or child.

The dosage forms described herein can also take the form ofpharmaceutical particles manufactured by a variety of methods, includingbut not limited to high-pressure homogenization, wet or dry ballmilling, or small particle precipitation (e.g., nGimat's NanoSpray).Other methods useful to make a suitable powder formulation are thepreparation of a solution of active ingredients and excipients, followedby precipitation, filtration, and pulverization, or followed by removalof the solvent by freeze-drying, followed by pulverization of the powderto the desired particle size. In one embodiment, the pharmaceuticalparticles have a final size of 3-1000 microns, such as at most 3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000microns. In another embodiment the pharmaceutical particles have a finalsize of 10-500 microns. In another embodiment the pharmaceuticalparticles have a final size of 50-600 microns. In another embodiment thepharmaceutical particles have a final size of 100-800 microns.

In another aspect, the disclosure provides a method of making aunit-dosage form described herein, comprising providing a glycantherapeutic (e.g., a glycan therapeutic described herein); formulatingthe glycan therapeutic into a unit-dosage form (e.g., a unit-dosage formdescribed herein), packaging the unit-dosage form, labelling thepackaged unit-dosage form, and/or selling or offering for sale thepackaged and labeled unit-dosage form.

The unit-dosage forms described herein may also be processed. In oneembodiment, the processing comprises one or more of: processing thedosage form into a pharmaceutical composition, e.g., formulating,combining with a second component, e.g., an excipient or buffer;portioning into smaller or larger aliquots; disposing into a container,e.g., a gas or liquid tight container; packaging; associating with alabel; shipping or moving to a different location. In one embodiment,the processing comprises one or more of: classifying, selecting,accepting or discarding, releasing or withholding, processing into apharmaceutical composition, shipping, moving to a different location,formulating, labeling, packaging, releasing into commerce, or selling oroffering for sale, depending on whether the predetermined threshold ismet. In some embodiments, the processed dosage forms comprise a glycantherapeutic described herein.

In some embodiments, the processing comprises one or more of: processingthe dosage form into a pharmaceutical composition, e.g., formulating,combining with a second component, e.g., an excipient or buffer;portioning into smaller or larger aliquots; disposing into a container,e.g., a gas or liquid tight container; packaging; associating with alabel; shipping or moving to a different location. In one embodiment,the processing comprises one or more of: classifying, selecting,accepting or discarding, releasing or withholding, processing into apharmaceutical composition, shipping, moving to a different location,formulating, labeling, packaging, releasing into commerce, or selling oroffering for sale, depending on the determination. In anotherembodiment, an oral dosage form is provided comprising a glycantherapeutic composition, wherein the oral dosage form is a syrup. Thesyrup can comprise about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% solid. The syrup can compriseabout 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% liquid, for example,water. The solid can comprise a glycan therapeutic composition. Thesolid can be, for example, about 1-96%, 10-96%, 20-96%, 30-96%, 40-96%,50-96%, 60-96%, 70-96%, 80-96%, or 90-96% glycan therapeuticcomposition. In another embodiment, a glycan therapeutic composition isformulated as a viscous fluid.

In one embodiment, the composition comprises a foaming component, aneutralizing component, or a water-insoluble dietary fiber. A foamingcomponent can be at least one member selected from the group consistingof sodium hydrogencarbonate, sodium carbonate, and calcium carbonate. Inone embodiment a neutralizing component can be at least one memberselected from the group consisting of citric acid, L-tartaric acid,fumaric acid, L-ascorbic acid, DL-malic acid, acetic acid, lactic acid,and anhydrous citric acid. In one embodiment a water-insoluble dietaryfiber can be at least one member selected from the group consisting ofcrystalline cellulose, wheat bran, oat bran, cone fiber, soy fiber, andbeet fiber. The formulation can contain a sucrose fatty acid ester,powder sugar, fruit juice powder, and/or flavoring material.

In some embodiments, the dosage forms are formulated to release thepharmaceutical compositions comprising glycan therapeutic preparationsin a specific region(s) of the GI tract, such as the small or the largeintestine. In some embodiments, the dosage forms are formulated torelease the pharmaceutical compositions comprising therapeutic glycanpreparations in a specific region(s) of the GI tract, such as the cecum,ascending colon, transverse colon, descending colon, sigmoid colon,and/or rectum.

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein is an enzyme-responsivedelivery system. For example, trypsin responsive polymers can be madeusing hydrogels that are crosslinked by peptides that are degraded bytrypsin. Trypsin is active in the small intestine. Trypsin-responsivedelivery systems can be used to target delivery of the pharmaceuticalglycan therapeutic compositions to the small intestine. In anotherexample, enzyme-digestible hydrogels consisting of poly(vinylpyrrolidone) crosslinked with albumin are degraded in the presence ofpepsin.

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein is a delivery device thatenables prolonged retention at a specific site in the GI tract. Forexample, a gastroretentive delivery system enables prolonged release ofthe pharmaceutical glycan therapeutic compositions to the stomach.Gastroretentive delivery may be used for the pharmaceutical glycantherapeutic compositions that modulate bacteria in the stomach or in theupper small intestine.

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein is a mucoadhesive deliverysystem that adheres to the mucosal surfaces of the stomach. They aretypically composed of polymers with numerous hydrogen-bonding groups,e.g., cross-linked polyacrylic acids, sodium carboxymethyl cellulose,sodium alginate, carrageenan, Carbopol 934P, or thiolated polycarbophil.

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein is an expanding deliverysystem that rapidly increases in size in the stomach, which slows itspassage through the pylorus. Such systems include systems that unfold inthe stomach. For example, geometric shapes such as tetrahedrons, rings,disks, etc. can be packed into a gelatin capsule. When the capsuledissolves, the shape unfolds. The systems can be composed of one or moreerodible polymer (e.g., hydroxypropyl cellulose), one or morenonerodible polymer (e.g., polyolefins, polyamides, polyurethanes). Theglycan therapeutic may then be dispersed within the polymer matrix. Theretention times can be fine-tuned by the polymer blend. Alternatively,devices made out of elastic polymers that are stable in the acidic pH ofthe stomach but dissolve in the neutral/alkaline conditions furtheralong the GI tract can be used. Such polymer formulations can preventintestinal obstruction when the device exits the stomach. Supramolecularpolymer gels crosslinked by hydrogen bonds between carboxyl groups mayalso be used, e.g. composed of poly(acryloyl 6-aminocaproic acid)(PA6ACA) and poly(methacrylic acid-co-ethyl acrylate) (EUDRAGIT L100-55). Other systems include swellable excipients, such as collagensponges. For example, a hydrogel matrix (e.g. a swellable core:polyvinyl pyrrolidone XL, Carbopol 934P, calcium carbonate) swells 2-50times in the stomach. Superporous hydrogel composites swell to hundredsof times their original volume in a few minutes. Some systems exploitgas generation to achieve expansion, e.g. carbon dioxide-generating,expandable systems that are surrounded by a hydrophilic membrane.

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein is a density-controlleddelivery system. These systems are designed to either float or sink ingastric fluids, which delays their emptying from the stomach. Forexample, high-density systems enable the device to settle to the bottomof the stomach, below the pylorus, and thus avoid stomach emptying.Other systems are low-density/floating systems. Such devices may, e.g.,comprise entrapped air in hollow chambers or may incorporate low-densitymaterials like fats, oils, or foam powder. Low density may be achievedthrough swelling, e.g. hydrocolloid containing capsules dissolve uponcontacting gastric fluid and the hydrocolloids swell to form a mucousbody. Alternative polymers include: chitosans, sodium alginate, andglycerol monooleate matrix. Low density may be achieved through gasgeneration. For example, tablets loaded with carbonate and optionallycitric acid generate carbon dioxide after contact with acidic aqueousmedia. The carbon dioxide generated is entrapped within the gellinghydrocolloid causing the system to float. Hydrocolloids includehydroxypropyl methylcellulose and Carbopol 934P.

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein employs a design to retain adevice in the small or large intestine. The location-specific nature ofthe device is provided by a specific triggering method, e.g. pH, enzyme,etc. These include systems designed for mucoadhesion and alsomicroneedle pills. Microneedle pills comprise a drug reservoir spikedwith microneedles that is encapsulated in a pH-responsive coating. Whenthe pill reaches the desired location in the GI tract and the coatingdissolves, the microneedles enable the pill to become stuck to thelining of the GI tract. In other embodiments, the microneedle pillscomprise a capsule that consists of two chemical compartments filledwith citric acid and sodium bicarbonate, respectively. As the pilldissolves in the digestive system, barriers between the two substanceserode, allowing them to mix and create a chemical reaction that pushesmicro-needles of saccharides through the outer layer of the capsule andinto the lining of the small intestine. The saccharide needles can befilled with drugs that are delivered into nearby blood vessels as thesaccharide is absorbed.

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein employs a pH sensitive polymercoating. For example, pH-dependent polymers (bi- or tri-phasic) can beinsoluble at low pH levels (e.g. acid resistance in the stomach, pH 1-2)and become increasingly soluble as pH rises, e.g. to about 5.5-6.2 inthe duodenum, to about pH 5.7 in the ascending colon, to about pH 6.4 inthe cecum, to about pH 6.6 in the transverse colon, to about pH 7.0 inthe descending colon, to about 7.2-7.5 in the ileum, or to about pH 7.5in the distal small intestine. In one example, TARGIT™ technology may beused for site-specific delivery of the pharmaceutical glycan therapeuticcompositions in the gastrointestinal (GI) tract. The system employspH-sensitive coatings onto injection-moulded starch capsules to targetthe terminal ileum and colon.

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein is a delayed release system ortime controlled release system. Such systems usually employ entericcoatings that may be combined with pH sensitive and time releasefunctions. For example, ETP (enteric coated time-release press coated)tablets may be used that are composed of three components: a glycantherapeutic-containing core tablet (rapid release function), apress-coated, swellable hydrophobic polymer layer (e.g. hydroxypropylcellulose layer (HPC), and a time release function. The duration of lagphase can be controlled either by weight or composition of polymer layerand an enteric coating layer (acid resistance function).

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein employs Eudragit® entericcoatings of tablets and capsules. Other suitable synthetic polymersinclude: Shellac, ethyl cellulose, cellulose acetate phthalate,hydroxypropylmethyl cellulose, polyvinyl acetate phthalate and polyglutamic acid coatings, such as poly-γ-glutamic acid (γ-PGA). Thesecoatings combine both mucoadhesive and pH-dependent release strategies.To enhance colon targeted delivery Eudragits® are methacrylicco-polymers with varying side group compositions that alter the pH atwhich they are soluble. For example, for Eudragit®-coated systems nosignificant drug release occurs in the stomach (e.g. at pH 1.4) and inthe small intestine (e.g. at pH 6.3), while significant drug release canbe seen at pH 7.8 in the ileocaecal region.

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein is a microbial-triggeredsystem, such as a polysaccharide based delivery system. Polysaccharidebased delivery systems contain biodegradable and mucoadhesive polymercoatings, including coatings of chitosan and pectin. Other suitablenatural polymers include, e.g., guar gum, inulin, cyclodextrin, dextran,amylase, chondrotin sulphate, and locust bean gum. These deliverysystems can be used to target the glycan therapeutic to the smallintestine. Coatings with naturally occurring polysaccharides like guargum, xanthan gum, chitosan, alginates, etc. are degraded by colonic gutmicrobiota, e.g. enzymes such as, xylosidase, arabinosidase,galactosidase etc. For example, CODES™ technology may be used to deliverthe pharmaceutical glycan therapeutic compositions. This system combinesthe polysaccharide coating with a pH-sensitive coating. In someembodiments, the system consists of a core tablet coated with threelayers of polymer coatings: The outer coating is composed of Eudragit L.This coating gets dissolved in the duodenum and exposes the nextcoating. The next coating is composed of Eudragit E. This layer allowsthe release of lactulose present in the inner core. The lactulose getsmetabolized into short chain fatty acids that lower the surrounding pHwhere the Eudragit E layer dissolves. The dissolving of Eudragit Eresults in the exposure of the glycan therapeutic. The bacteria presentin the colon are responsible for the degradation of polysaccharides thatare released from the core tablet. The degradation of polysaccharidesmay result in organic acids formation that lowers the pH of the contentssurrounding the tablet.

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein is a pressure-controlleddelivery system. The system employs the fact that higher pressures areencountered in the colon than in the small intestine. For example, forethylcellulose systems that are insoluble in water, the release ofglycan therapeutics occurs following disintegration of a water-insolublepolymer capsule as a result of pressure in the lumen of the colon. Therelease profile may be adjusted by varying the thickness of theethylcellulose, the capsule size and/or density of the capsule.

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein is a pulsatile colon targeteddelivery system. For example, the system can be a pulsincap system. Thecapsule which is employed comprises a plug that is placed in the capsulethat controls the release of the glycan therapeutic. A swellablehydrogel (e.g. hydroxyl propyl methyl cellulose (HPMC), poly methylmethacrylate or polyvinyl acetate) seals the drug content. When thecapsule gets in contact with a fluid the plug is pushed off from thecapsule and the glycan therapeutic is released. The release profile canbe controlled by varying the length and/or point of intersection of theplug with the capsule body. Another system is a port system. The capsulebody is enclosed in a semi-permeable membrane. The insoluble plugconsists of an osmotically active agent and the glycan therapeutic. Whenthe capsule gets in contact with a fluid the semi-permeable membranepermits inflow of the fluid which increases pressure in the capsulebody. This leads to an expelling of the plug and release of the glycantherapeutic.

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein is an osmotically controlledcolon targeted delivery system. An exemplary system, OROS-CT, consistsof osmotic units (up to 5 or 6 push pull units) encapsulated in a hardgelatin capsule. The push pull units are bilayered with outer entericimpermeable membrane and inner semi-permeable membrane. The internal,central part of the push pull consists of the drug layer and push layer.The glycan therapeutic is released through the semi-permeable membrane.The capsule body enclosing the push pull units is dissolved immediatelyafter administration. In the GI tract the enteric impermeable membraneprevents water absorption. The enteric coating is dissolved in smallintestine (higher pH, >7), water enters the unit through thesemi-permeable membrane causing push layer to swell and force out theglycan therapeutic.

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein is “smart pill” which can beused to release the glycan therapeutic just before reaching theileocecal valve.

In some embodiments, the dosage form for the pharmaceutical glycantherapeutic compositions described herein is a rectally administeredformulation. For example, enemas introduce a pharmaceutical glycantherapeutic composition in liquid formulation into the rectum. Thevolume administered is typically less than 10 mL. Suppositoriesintroduce a pharmaceutical glycan therapeutic composition into therectum. Suppositories are solid dosage forms that melt or dissolve wheninserted into the rectum, releasing the glycan therapeutics. Typicalexcipients for suppository formulations include cocoa butter,polyethylene glycols, and agar.

Generation of Polyphenols, Synthesis and Preparation of Extracts

Provided herein are pharmaceutical compositions comprising glycantherapeutic preparations and polyphenol preparations. In someembodiments, the pharmaceutical compositions comprising glycantherapeutic preparations comprise at least one polyphenol. In someembodiments, the pharmaceutical compositions comprising glycantherapeutic preparations comprise a plurality of polyphenols.

The preparations of glycan therapeutics and the preparations ofpolyphenols may be generated separately. For example, the preparation ofglycan therapeutics may be synthesized and the preparation ofpolyphenols may be extracted as described herein. In another example,the preparation of glycan therapeutics may be synthesized and thepreparation of polyphenols may also be synthesized as described herein.In yet another example, the preparation of glycan therapeutics may besynthesized and the preparation of polyphenols may comprise a pluralityof polyphenols that was extracted from a source and a plurality that wassynthesized.

In some embodiments, preparations of polyphenols are generated fromextracts containing the polyphenols. In other embodiments, thepreparations of polyphenols comprise polyphenols that are synthesized.

Pharmaceutical compositions and medical foods may be generated by mixingthe preparation of glycan therapeutics with the preparation ofpolyphenols by any suitable method known in the art. In someembodiments, the preparations are mixed in a 0.000000001:1,0.00000001:1, 0.0000001:1, 0.000005:1, 0.00001:1, 0.0001:1. 0.001:1,0.01:1, 0.1:1, 0.5:1, 1:1, 1:2, 1:5, 1:10, 1:100, 1:1,000, 1:10,000,1:100,000, 1:1000,000 (v/v), (w/w), (w/v) or molar ratio(glycan:polyphenol, polyphenol:glycan).

In some embodiments, the preparations of polyphenols are extracted fromplants, plant parts, plant cells, or plant products. Examples of plantparts are but not limited to bark, flower, petal, stem, stalk, tuber,root, fruit, berry, seed, nut, leaf. Examples of plant products includebut are not limited to pomace juice, pulp, skin, mash, paste, andslurry. Examples of plants may include but are not limited to blueberry,cranberry, grape, peach, plum, pomegranate, soy, red wine, black tea,green tea. In some embodiments, the polyphenols are extracted frommultiple plants, plant parts, or plant products. In some embodiments thepolyphenol extract is combined from multiple plants, plant parts, orplant products.

Polyphenols may be extracted from any suitable source, such as, e.g.foods rich in polyphenols, including, but not limited to, cloves,peppermint, star anise, cocoa powder, oregano, celery seed, blackchokeberry, dark chocolate, flaxseed meal, black elderberry, chestnut,common sage, rosemary, spearmint, common thyme, lowbush blueberry,blackcurrant, capers, black olive, highbush blueberry, hazelnut, pecannut, soy flour, plum, green olive, sweet basil, curry, powder, sweetcherry, globe artichoke heads, blackberry, roasted soybean, milkchocolate, strawberry, red chicory, red raspberry, coffee, filter,ginger, whole grain hard wheat flour, prune, almond, black grape, redonion, green chicory, common thyme, fresh, refined maize flour, soy,tempeh, whole grain rye flour, apple, spinach, shallot, lemon verbena,black tea, red wine, green tea, soy yogurt, yellow onion, soy meat,whole grain wheat flour, pure apple juice, pure pomegranate juice,extra-virgin olive oil, black bean, peach, pure blood orange juice,cumin, pure grapefruit juice, white bean, Chinese cinnamon, pure blondorange juice, broccoli, redcurrant, soy tofu, pure lemon juice, wholegrain oat flour, apricot, caraway, refined rye flour, asparagus, walnut,potato, Ceylan cinnamon, parsley, nectarine, curly endive, marjoram, redlettuce, chocolate beverage with milk, quince, endive (escarole), soymilk, pure pummelo juice, rapeseed oil, pear, soybean sprout, greengrape, carrot, vinegar, soy cheese, white wine, and rosé wine.

In some embodiments, polyphenols may be extracted from plant juice. Insome embodiments, the plant juice is the juice of a blueberry,blackberry, raspberry, hockenberry, gooseberry, boysenberry, acai berry,baneberry, barberry, bearberry, bilberry, chokeberry, bunchberry,buffalo berry, chokecherry, cowberry, elderberry, cranberry, dew berry,currant, farkleberry, goji berry, gooseberry, grape, holly berry,huckleberry, ivy berry, june berry, juniper berry, lingonberry, loganberry, mistletoe berry, nannyberry, Oregon grape, persimmon, pokeberry,privet berry, salmonberry, strawberry, sugarberry, tayberry,thimbleberry, white mulberry, red mulberry, black mulberry, wineberry,wintergreen, yew berry, or young berry.

In some embodiments, polyphenols may be extracted from plants or planttissue that have been modified, bred, engineered, or otherwise changed,e.g., to alter the composition or quantity of polyphenol contents. Insome embodiments, the plants or plant tissue is treated, subjected to,or contacted with a polyphenol-inducing agent (e.g. a chemical agent orradiation, such as UV) prior to or after harvesting or isolating of theplant or plant tissue to induce or stimulate the production ofpolyphenols by the plant or plant tissue or to increase the relativeamount of polyphenols in the plant or plant tissue, when e.g. comparedto a non-treated plant or tissue or a plant or tissue that has not beensubjected to or contacted with the polyphenol-inducing agent.

In some embodiments, the pharmaceutical compositions of glycantherapeutics comprise preparations of polyphenols that can be extractedby any method known in the art. For example, the extraction method maycomprise one or more of the following steps: i) drying the source; ii)milling, grinding, crushing, blending, or otherwise homogenizing thesource; iii) extracting the polyphenols from the source, e.g. using asolvent.

The source material may optionally be pretreated with enzymes or treatedwith enzymes during extraction. Examples of enzymes include but are notlimited to pectinolytic and cell-wall polysaccharide degrading enzymes.

The solvent may be any suitable solvent known in the art. For example,the solvent can be an organic solvent, such as but not limited to,methanol, ethanol, acetone, and ethyl acetate optionally an aqueoussolvent (comprising water). If desired, the solvent may include organicacids, such as but not limited to trifluoroacetic acid, formic acid,acetic acid, citric acid, hydrochloric acid, tartaric acid, sulfuricacid, or phosphoric acid. The solvent may be a mixture of an organicsolvent and an acid in any suitable ratio. The solvent may includesupercritical CO₂.

The extraction may be carried out between 0 and 100° C. Extractionmethods include: maceration and soxhlet extraction, rotary evaporation,microwave-assisted extraction, ultrasound-assisted extraction,subcritical water extraction, supercritical fluid extraction,pressurized fluid extraction, pressured liquid extraction, andaccelerated solvent extraction.

In some embodiments, the extraction may be performed multiple times uponthe same source material. If desired, multiple extractions from thesource material may be combined. In other embodiments, multipleextractions from different source materials may be combined.

Purification and fractionation of the polyphenol extract may beaccomplished by any suitable method known in the art, including, but notlimited to: i) sequential extraction or liquid-liquid partitioning, ii)solid phase extraction, iii) countercurrent chromatography, and iv)centrifugation. For sequential extraction, the crude extract may bewashed with non-polar solvents to remove lipids. Examples of non-polarsolvents are but not limited to hexane, dichloromethane, and chloroform.For solid phase extraction, the crude extract may be washed over a solidphase binding substrate to separate polyphenols substituents and/orremove sugars. In some embodiments, water-soluble constituents such assugar and organic acids are removed with acidified water. Examples ofsolid phase binding substrates are but not limited to C18, AmberliteXAD-2, XAD-7, XAD-16, Oasis HLB, Silica-based C8, copolymer-based HLB,PH, ENV+, RP-C18, Toyopearl, LH-20, polyamide resin, and MCX. Ifdesired, the polyphenols may be further fractionated by adjusting theeluent solvent and solvent pH. Examples of eluents include but are notlimited to ethanol, methanol, acetone, and water, and any combinationthereof. In some embodiments, phenolic acids are eluted with water. Insome embodiments, nonpolymeric phenols are eluted with acidified ethylacetate. In some embodiments, polymeric phenols are eluted with acombination of water, acetone, ethanol, and/or methanol. In someembodiments, procyanidins are eluted with acetone and water.

As one example, proanthocyanidins may be isolated from grape berryskins. Grape skins can be collected after the juice is squeezed from thegrape berries and removed. A suitable solvent, e.g. an acetone/watermixture may be used to extract polyphenols from the grape berry skins.The solvent is then removed. Aqueous phase extraction may be carriedout, e.g. with cloroform and the extracts can optionally be freezedried. The resulting powder may be purified using adsorptionchromatography. The proanthocyanidins can be washed and eluted with asolvent (e.g. methanol, acetone) and trifluoroacetic acid. The solventis removed and the acqueous phase optionally freeze dried to generateproanthocyanidin powders.

Various methods can be used to characterize the resultingproanthocyanidin mixtures. Acid-catalysis in the presence of excessphloroglucinol may be used to determine subunit composition, conversionyield, and mean degree of polymerization. Mass distribution ofpolyphenols is determined with mass spectrometry. The mass spectrometrymethod consists of dissolution of polyphenols in a suitable solvent(e.g. methanol/acetonitrile) and infusion into the electrosprayer. Gelpermeation chromatography can provide spectra of eluting peaks and isrun with two columns in series (for example, TSKgel G3000 Hxl particlesize 6 um followed by G2500 Hxl particle size 5 um, both 300×7.8 mmi.d.), carried out under isocratic conditions with the mobile phase asdimethylformamide Pigmented proanthocyanidins can be characterized withUV-Vis spectrophotometry. Finally, elemental analysis for C, H, and Ncan be done by loading powder sample into a tin cup and running analysisusing an elemental analyzer, such as, e.g., a Carlo Erba EZ 1108.

In some embodiments, the compositions described herein comprisesynthetic polyphenols. Polyphenols may not only be derived from suitableplant sources (e.g. plant extracts) but can be synthesized. Polyphenolsmay be synthesized via a suitable combination of chemical, biochemical,or biotechnological methods known in the art. In some embodiments, thepolyphenols are extracted from natural sources and subsequently modifiedvia chemical, biochemical, or biotechnological methods. In someembodiments, the synthesized or modified polyphenol chemical structuresare not found in nature. Examples of chemical modifications include butare not limited to methylation, hydroxylation, prenylation,glycosylation, dimzerization, polymerization. Examples of glycosylationinclude but are not limited to glucosides, galactosides, arabinosides,rhamnosides.

Methods for chemical synthesis of polyphenols are known in the art andare described, e.g., in Quideau S et al. “Plant Polyphenols: ChemicalProperties, Biological Activities, and Synthesis”, Angewandte Chemie2011, 50, 586-621. In some embodiments, the polyphenols are synthesizedor modified via biotechnological methods, e.g. using enzymes to catalyzesuitable reactions. The reactions may occur within cells (e.g. inbacteria, yeast, plant cells) or in extracts or lysates in or obtainedfrom e.g. from bacteria, yeast, or plant cells. In some embodiments,specific enzymes are isolated and used in suitable buffer systems andunder suitable reaction conditions. Methods for biotechnologicalsynthesis of polyphenols are known in the art and are described, e.g.,in Cress B et al. “Isoflavonoid Production By Genetically EngineeredMicroorganisms”, Natural Products. Springer-Verlag Berlin Heidelberg,2013. 1647-1681; Trantas E A et al. “When Plants Produce Not Enough OrAt All: Metabolic Engineering Of Flavonoids In Microbial Hosts”,Frontiers in Plant Science 2015, 6.

Polyphenols may be quantified or measured by any suitable method. Insome embodiments, known concentrations of a reference standard (e.g. apolyphenol or plurality of polyphenols) are used for comparison in ameasurement. In some embodiments, total polyphenols are quantified viathe Folin-Denis method, Folin-Ciocalteau method, permanganate titration,colorimetry with iron salts, HPLC, precipitation of substrates, orelectromagnetic absorbance.

In some embodiments, polyphenol classes are quantified. For example,anthocyanins may be quantified using electromagnetic absorbance betweenwavelength 490 and 550 nM at one or multiple pH. Proanthocyanidins canbe quantified using coloriometric methods, substrate precipitation, orprotein binding assays, or a combination thereof. In another example,tannins can be quantified using potassium iodide, rhodanine, or sodiumnitrite, or protein binding assays, or a combination thereof. In someembodiments, gas chromatography is used for the separation andquantification of polyphenols or a plurality of polyphenols. If desired,the polyphenols may be modified prior to gas chromatography, e.g. tomake the polyphenols more volatile.

Alternatively or in addition, the polyphenols are quantified via HPLC,optionally using various solid supports and mobile phases. Polyphenolsmay also be quantified via mass spectrometry (MS). In some embodiments,the polyphenols are quantified with HPLC-MS optionally using varioussolid supports and mobile phases. In some embodiments, the antioxidantcapacity of polyphenols is measured. For example, the antioxidantcapacity of polyphenols can be measured via Trolox equivalentantioxidant capacity assay, oxygen radical absorbance capacity assay,total radical-trapping antioxidant parameter assay, ferric ion reducingantioxidant power assay, cupric ion reducing antioxidant capacity assay,or a combination thereof.

Other methods for phenolic extraction, purification, analysis andquantification are known in the art and are described, e.g., in Dai J.and Mumper R J, “Plant Phenolics: Extraction, Analysis and TheirAntioxidant and Anticancer Properties”, Molecules 2010, 15(10),7313-7352.

In some embodiments, polyphenols can be extracted and/or concentratedusing proteins from various sources and optionally varying the pH, e.g.,described in Raskin et al., U.S. patent publication No. 20140328997.

In some embodiments, the yield of extracted polyphenols is μg/kg sourcematerial. In some embodiments, the yield of extracted polyphenols ismg/kg source material. In some embodiments the yield of extractedpolyphenols is g/kg source material.

In some embodiments, the preparation of polyphenols includes flavonoids.In some embodiments, the flavonoids are anthocyanins, anthocyanidins,chalcones, dihydrochalcones, dihydroflavonols, flavanols, flavan-3-ols,flavanones, flavones, flavonols, isoflavonoids, proanthocyanidins,condensed tannins, non-hydrolyzable tannins. In some embodiments theflavonoids are monomers. In some embodiments the flavonoids are dimers.In some embodiments the flavonoids are polymers. In some embodiments theflavonoids are chemically modified. Examples of chemical modificationsare but not limited to methylation, hydroxylation, prenylation,glycosylation. Examples of glycosylation are but not limited toglucosides, galactosides, arabinosides, rhamnosides.

In some embodiments, the preparation of polyphenols includeshydrolyzable tanins, phlorotannins, lignans, alkymethoxyphenols,alkyphenols, curcumoids, furanocoumarins, hydroxybenzaldehydes,hydroxybenzoketones, hydroxycinnamaldehyde, hydroxycoumarins,hydroxyphenylpropenes, methoxyphenols, naphtoquinones, phenolicterpenes, tyrosols, arbutin, catechol, pyrocatechol, resorcinol,coumestrol, phenol, phlorin, pyrogallol, phloroglucinol, salvianolicacid, hydroxybenzoic acid, hydroxycinnamic acid, hydroxyphenylaceticacid, hydroxyphenylpropanic acid, hydroxyphenylpentanoic acid,stilbenes. In some embodiments the polyphenols are monomers. In someembodiments the polyphenols are dimers. In some embodiments thepolyphenols are polymers. In some embodiments the polyphenols arechemically modified. Examples of chemical modifications are but notlimited to methylation, hydroxylation, prenylation, glycosylation.Examples of glycosylation are but not limited to glucosides,galactosides, arabinosides, rhamnosides.

In some embodiments, the extracts comprise a plurality of one or more(e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90,100 or more) polyphenols listed in Table 5. In some embodiments, thepharmaceutical compositions comprising glycan therapeutics describedherein comprise a plurality of one or more (e.g. 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more) polyphenols listedin Table 5.

Kits

Kits also are contemplated. For example, a kit can comprise unit dosageforms of the pharmaceutical glycan therapeutic composition, and apackage insert containing instructions for use of the glycan therapeuticin treatment of a gastrointestinal disorder or condition. The kitsinclude a pharmaceutical glycan therapeutic composition in suitablepackaging for use by a subject in need thereof. Any of the compositionsdescribed herein can be packaged in the form of a kit. A kit can containan amount of a pharmaceutical glycan therapeutic composition (optionallyadditionally comprising a prebiotic substance, a probiotic bacterium,and/or a second therapeutic agent) sufficient for an entire course oftreatment, or for a portion of a course of treatment. Doses of apharmaceutical glycan therapeutic composition can be individuallypackaged, or the pharmaceutical glycan therapeutic composition can beprovided in bulk, or combinations thereof. Thus, in one embodiment, akit provides, in suitable packaging, individual doses of a glycantherapeutic composition that correspond to dosing points in a treatmentregimen, wherein the doses are packaged in one or more packets.

In one embodiment, the pharmaceutical glycan therapeutic composition canbe provided in bulk in a single container, or in two, three, four, five,or more than five containers. For example, \each container may containenough of a pharmaceutical glycan therapeutic composition for aparticular week of a treatment program that runs for a month. If morethan one bulk container is provided, the bulk containers can be suitablypackaged together to provide sufficient pharmaceutical glycantherapeutic composition for all or a portion of a treatment period. Thecontainer or containers can be labeled with a label indicatinginformation useful to the subject in need thereof or the physicianperforming the treatment protocol, such as, e.g. dosing schedules.

The pharmaceutical glycan therapeutic composition can be packaged withother suitable substances, such as probiotic bacteria, prebioticsubstances or other substances, as described herein. The other substanceor substances can be packaged separately from the pharmaceutical glycantherapeutic composition, or mixed with the pharmaceutical glycantherapeutic composition, or combinations thereof. Thus, in oneembodiment, kits include a dosage form containing all the ingredientsintended to be used in a course of treatment or a portion of a course oftreatment, e.g., a pharmaceutical glycan therapeutic composition andoptionally buffers, excipients, etc., a probiotic, prebiotic or atherapeutic agent. In one embodiment, a pharmaceutical glycantherapeutic composition is packaged in one package or set of packages,and additional components, such as probiotic bacteria, prebiotics, andtherapeutic agents are packaged separately from the pharmaceuticalglycan therapeutic composition.

Kits can further include written materials, such as instructions,expected results, testimonials, explanations, warnings, clinical data,information for health professionals, and the like. In one embodiment,the kits contain a label or other information indicating that the kit isonly for use under the direction of a health professional. The containercan further include scoops, syringes, bottles, cups, applicators orother measuring or serving devices.

Methods of Modulating Microbial Taxa, Genomic and Functional States ofthe Microbiome

Provided herein are method for modulating the abundance of bacterialtaxa (e.g. 1, 2, 3, 4, 5 or more taxa) in a human subject'sgastrointestinal microbiota. These methods include administering to thehuman subject a pharmaceutical composition comprising a glycantherapeutic preparation in an amount effective to modulate the abundanceof the taxa. The abundance of a bacterial taxa may increase relativelyto other taxa (or relative from one point in time to another) when theglycan therapeutic is administered and the increase can be at least a5%, 10%, 25% 50%, 75%, 100%, 250%, 500%, 750% increase or at least a1000% increase. The abundance of a bacterial taxa may also decreaserelative to other taxa (or relative from one point in time to another)when the glycan therapeutic is administered and the decrease can be atleast a 5%, 10%, 25% 50%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99%decrease, or at least a 99.9% decrease. In some embodiments, a dysbiosishas shifted the microbiota and has increased one or more non-desiredtaxa and/or increased one or more desired taxa. Administration of theglycan therapeutic can modulate the abundance of the desired and/ornon-desired bacterial taxa in the subject's gastrointestinal microbiota,thereby treating the dysbiosis.

In some embodiments, the glycan therapeutic is capable of modulating(e.g. increasing or decreasing) the growth of one or more bacterium,such as, e.g., those that belong to genera Bacteroides, Odoribacter,Parabacteroides, Alistipes, Blautia, Clostridium, Coprococcus, Dorea,Eubacterium, Lachnospira, Roseburia, Ruminococcus, Faecalibacterium,Oscillospira, and Subdoligranulum which can be found in the GI tract. Insome embodiments, the glycan therapeutic is capable of modulating (e.g.increasing or decreasing) the growth of one or more bacterium, such as,e.g., those that are thought to be associated with a healthygastrointestinal state, such as, for example, one or more of the genusAkkermansia, Anaerofilum, Bacteroides, Blautia, Bifidobacterium,Butyrivibrio, Clostridium, Coprococcus, Dialister, Dorea, Fusobacterium,Eubacterium, Faecalibacterium, Lachnospira, Lactobacillus,Phascolarctobacterium, Peptococcus, Peptostreptococcus, Prevotella,Roseburia, Ruminococcus, and Streptococcus, and/or one or more of thespecies Akkermansia municiphilia, Christensenella minuta, Clostridiumcoccoides, Clostridium leptum, Clostridium scindens, Dialister invisus,Eubacterium rectal, Eubacterium eligens, Faecalibacterium prausnitzii,Streptococcus salivarius, and Streptococcus thermophilus.

In some embodiments, the glycan therapeutic is capable of modulating(e.g. increasing or decreasing) the growth of at least two bacterialtaxa selected from Prevotella, Akkermansia, Bacteroides, Clostridium(Erysipelotrichaceae), Clostridium (Clostridiaceae), Bifidobacterium,Aggregatibacter, Clostridium (Peptostreptococcaveae), Parabacteroides,Lactobacillus, and Enterococcus. Exemplary glycan therapeutics includeglu100, ara100, glu50gal50, and glu33gal33fuc33, In some embodiments,the glycan therapeutic is capable of modulating the growth of the twobacterial taxa: Akkermensia and Blautia. An exemplary glycan therapeuticis xyl100.

In some embodiments, the glycan therapeutics drive selective changes inboth the composition and activity (or function) of the gastrointestinalmicrobiota, thereby conferring health benefits upon the host. In someembodiments, the glycan therapeutic is a selective substrate for one ora limited number of potentially beneficial bacteria that reside in theGI tract, stimulating their growth and/or metabolic activity. In someembodiments, the glycan therapeutic is capable of altering thecomposition of gastrointestinal microbiota to a composition higher orlower in specific bacteria. In some embodiments, the glycan therapeuticselectively stimulates the growth and/or selective activity ofgastrointestinal bacteria associated with health and well-being. In oneexample, the glycan therapeutic compositions described herein decreasethe abundance or relative number or density of a pathogenic bacterium.

The relationship between microbiota and their host is not merelycommensal (a non-harmful coexistence), but in many cases a symbioticrelationship. Though subjects can survive without microbiota, themicroorganisms perform a variety of useful functions, such as fermentingunused energy substrates, training the immune system, preventing growthof pathogenic bacteria, regulating the development of the gut, producingvitamins for the host (such as biotin and vitamins) (See, e.g.,Dominguez-Bello M G and Blaser M J, 2008 Microbes Infect, 10(9):1072-1076). Common gastrointestinal bacterial taxa include generaBacteroides, Odoribacter, Parabacteroides, Alistipes, Blautia,Clostridium, Coprococcus, Dorea, Eubacterium, Lachnospira, Roseburia,Ruminococcus, Faecalibacterium, Oscillospira, and Subdoligranulum. Somebacterial genera and species are thought to be associated with a healthystate of the GI tract, such as, e.g., the genus Akkermansia,Anaerofilum, Bacteroides, Blautia, Bifidobacterium, Butyrivibrio,Clostridium, Coprococcus, Dialister, Dorea, Fusobacterium, Eubacterium,Faecalibacterium, Lachnospira, Lactobacillus, Phascolarctobacterium,Peptococcus, Peptostreptococcus, Prevotella, Roseburia, Ruminococcus,and Streptococcus, and/or the species Akkermansia municiphilia,Christensenella minuta, Clostridium coccoides, Clostridium leptum,Clostridium scindens, Dialister invisus, Eubacterium rectal, Eubacteriumeligens, Faecalibacterium prausnitzii, Streptococcus salivarius, andStreptococcus thermophilus.

However, in certain conditions, pathogenic species and pathobionts whichare capable of causing disease, e.g. by inducing an infection and/orinflammation and/or bacteria associated with a disease state withoutnecessarily being a causative agent, are present in the niche. In someembodiments, disease-associated bacteria, pathobionts or pathogens thatmay be modulated by the glycan therapeutics described herein areselected from the group consisting of the genera Bilophila,Campylobacter, Candidatus, Citrobacter, Clostridium, Collinsella,Desulfovibrio, Enterobacter, Enterococcus, Escherichia, Fusobacterium,Haemophilus, Klebsiella, Lachnospiraceae, Peptostreptococcus,Porphyromonas, Portiera, Providencia, Pseudomonas, Salmonella, Shigella,Staphylococcus, Streptococcus, Vibrio, and Yersinia.

In some embodiments, disease-associated bacteria, pathobionts orpathogens that may be modulated by the glycan therapeutics describedherein are selected from the group consisting of the species Bilophilawadsworthia, Campylobacter jejuni, Citrobacter farmer, Clostridiumdifficile, Clostridium perfringens, Clostridium tetani, Collinsellaaerofaciens, Enterobacter hormaechei, Enterococcus faecalis,Enterococcus faecium, Escherichia coli, Fusobacterium varium,Fusobacterium nucleatum, Haemophilus parainfluenzae, Klebsiellapneumonia, Peptostreptococcus stomatis, Porphyromonas asaccharolytica,Pseudomonas aeruginosa, Salmonella bongori, Salmonella enteric, Shigellaboydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei,Staphylococcus aureus, Streptococcus infantarius, Vibrio cholera, andYersinia enterocolitica.

In some embodiments, disease-associated bacteria, pathobionts orpathogens that may be modulated by the glycan therapeutics describedherein may reside predominantly in one or more specific regions of theGI tract.

For example, the following disease-associated bacteria, pathobionts andpathogens reside predominantly in the large intestine (colon): Listeria,Entamoeba histolytica, Balantidium coli, Basidiobolus ranarum,Trypanosoma cruzi, Clostridium botulinum, Fasciola hepatica, Histoplasmacapsulatum, Rotavirus, Schistosoma mansoni, Schistosoma japonicum, andSchistosoma mekongi, Shigella, Brachyspira aalborgi, Serpulinapilosicoli, Trichuris trichiura, and Yersinia enterocolitica.

The following disease-associated bacteria, pathobionts and pathogensreside predominantly in the small intestine: Vibrio, Yersiniaenterocolitica, Yersinia pseudotuberculosis, Clostridium perfringens,Capillaria philippinensis, Cryptosporidium parvum, Cyclosporacayetanensis and CMV virus.

The following disease-associated bacteria, pathobionts and pathogensreside predominantly in the large and small intestine: Campylobacter andSalmonella.

In another example, the following disease-associated bacteria,pathobionts and pathogens reside predominantly in the stomach: CMVvirus, Bacillus anthracis, Candida, Cryptosporidium, EBV (Epstein-Barrvirus), Giardia lamblia, Helicobacter pylori, Helicobacter felis,Helicobacter fennelliae, Helicobacter cinaedi, Mycobacterium avium,Herpes varicella zoster, Histoplasma, and Toxoplasma.

A healthy microbial community protects the host, e.g., by enhancing theintestinal barrier, by competitive exclusion of potential pathogens ordisease-associated bacteria, and by growth inhibition of bacterialpathogens and disease-associated bacteria. A healthy bacterial communitymay exert direct antibacterial effects on pathogens anddisease-associated bacteria through production of antibacterialsubstances, including bacteriocins and acid (Cotter P D, et al. 2005 NatRev, 3:777-788; Servin A L, 2004 FEMS Microbiol Rev, 28: 405-440). Theantibacterial substances exert their effects alone or synergistically toinhibit the growth of pathogens or disease-associated bacteria. Ahealthy bacterial community may decrease adhesion of both pathogens andtheir toxins to gastrointestinal lining.

In some embodiments, the glycan therapeutic modulates (e.g. increases ordecreases) the growth of one or more bacterial taxa residing in the GItract, such as, e.g., those that belong to genera Bacteroides,Odoribacter, Parabacteroides, Alistipes, Blautia, Clostridium,Coprococcus, Dorea, Eubacterium, Lachnospira, Roseburia, Ruminococcus,Faecalibacterium, Oscillospira, and Subdoligranulum which can be foundin the GI tract. In some embodiments, the glycan therapeutic modulates(e.g. increases or decreases) the growth of one or more bacterial taxa,such as those that are thought to be associated with a healthygastrointestinal state, e.g., one or more of the genus Akkermansia,Anaerofilum, Bacteroides, Blautia, Bifidobacterium, Butyrivibrio,Clostridium, Coprococcus, Dialister, Dorea, Fusobacterium, Eubacterium,Faecalibacterium, Lachnospira, Lactobacillus, Phascolarctobacterium,Peptococcus, Peptostreptococcus, Prevotella, Roseburia, Ruminococcus,and Streptococcus, and/or one or more of the species Akkermansiamuniciphilia, Christensenella minuta, Clostridium coccoides, Clostridiumleptum, Clostridium scindens, Dialister invisus, Eubacterium rectal,Eubacterium eligens, Faecalibacterium prausnitzii, Streptococcussalivarius, and Streptococcus thermophilus. In some embodiments, theglycan therapeutic modulates (e.g. increases or decreases) the growth ofone or more bacterial taxa, such as taxa of the phylum Verrucomicrobia,e.g., those of the genus Akkermansia.

In some embodiments, modulates (e.g. increases or decreases) the growthof one or more bacterial taxa predominantly residing in the smallintestine. For example, the glycan therapeutic modulates one or more (2,3, 4, 5, 6, 7, 8, 9, 10 or more) bacterial taxa that residepredominantly in the small intestine, such as, e.g. Actinobacteria,Firmicutes (Bacilli, Clostridia), and Proteobacteria(Alphaproteobacteria, Betaproteobacteria). In some embodiments, theglycan therapeutic modulates one or more (2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) bacterial taxa that reside predominantly in the small intestineselected from the genera: Cryocola, Mycobacterium, Enterococcus,Lactococcus, Streptococcus, Turicibacter, Blautia, Coprococcus,Holdemania, Pseudoramibacter Eubacterium, Agrobacterium, Sphingomonas,Achromobacter, Burkholderia, and Ralstonia.

In some embodiments, the glycan therapeutic modulates (e.g. increases ordecreases) the growth of one or more bacterial taxa predominantlyresiding in the large intestine. For example, the glycan therapeuticmodulates one or more (2, 3, 4, 5, 6, 7, 8, 9, 10 or more) bacterialtaxa that reside predominantly in the large intestine, such as, e.g.Bacteroidetes, Firmicutes (Clostridia), Verrucomicrobia, andProteobacteria (Deltaproteobacteria). In some embodiments, the glycantherapeutic modulates one or more (2, 3, 4, 5, 6, 7, 8, 9, 10 or more)bacterial taxa that reside predominantly in the large intestine selectedfrom the genera: Bacteroides, Butyricimonas, Odoribacter,Parabacteroides, Prevotella, Anaerotruncus, Phascolarctobacterium,Ruminococcus, Bilophila, and Akkermansia.

In some embodiments, the glycan therapeutic modulates (e.g. increases ordecreases) the growth of one or more bacterial taxa predominantlyresiding in the cecum, such as, e.g. Actinobacteria, Bacteroides,Bacilli, Clostridia, Mollicutes, Alpha Proteobacteria, andVerrucomicrobia.

In some embodiments, the glycan therapeutic modulates (e.g. increases ordecreases) the growth of one or more bacterial taxa predominantlyresiding in the ascending colon, such as, e.g. Actinobacteria,Bacteroides, Bacilli, Clostridia, Fusobacteria, Beta Proteobacteria,Delta/Epsilon Proteobacteria, Gamma Proteobacteria, and Verrucomicrobia.

In some embodiments, the glycan therapeutic modulates (e.g. increases ordecreases) the growth of one or more bacterial taxa predominantlyresiding in the traverse colon, such as, e.g. Actinobacteria,Bacteroides, Clostridia, Mollicutes, Fusobacteria, and GammaProteobacteria.

In some embodiments, the glycan therapeutic modulates (e.g. increases ordecreases) the growth of one or more bacterial taxa predominantlyresiding in the descending colon, such as, e.g. Bacteroides, Clostridia,Mollicutes, Fusobacteria, Delta/Epsilon Proteobacteria andVerrucomicrobia.

In some embodiments, the glycan therapeutic modulates (e.g. increases ordecreases) the growth of one or more bacterial taxa predominantlyresiding in the sigmoid colon, such as, e.g. Actinobacteria,Bacteroides, Bacilli, Clostridia, Mollicutes, Alpha Proteobacteria, BetaProteobacteria, and Verrucomicrobia.

In some embodiments, the glycan therapeutic modulates (e.g. increases ordecreases) the growth of one or more bacterial taxa predominantlyresiding in the rectum, such as, e.g. Bacteroides, Clostridia,Mollicutes, Alpha Proteobacteria, Gamma Proteobacteria, andVerrucomicrobia.

In some embodiments, the glycan therapeutics modulate (e.g.stimulate/increase or suppress/decrease) the growth of one or more (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 150, 200, or morethan 200) endogenous commensal microbial taxa or exogenouslyadministered probiotic bacterial taxa of various genera including, e.g.Alistipes, Akkermansia, Anaerofilum, Bacteroides, Blautia,Bifidobacterium, Butyrivibrio, Clostridium, Coprococcus, Dialister,Dorea, Fusobacterium, Eubacterium, Faecalibacterium, Lachnospira,Lactobacillus, Odoribacter, Oscillospira, Parabacteroides,Phascolarctobacterium, Peptococcus, Peptostreptococcus, Prevotella,Roseburia, Ruminococcus, and Streptococcus and Subdoligranulum.

In some embodiments, the glycan therapeutics modulate (e.g.stimulate/increase or suppress/decrease) the growth of one or more (e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 150, 200, or morethan 200) endogenous commensal or symbiotic microbial taxa orexogenously administered probiotic bacterial taxa of various generaincluding, but not limited to, bacterial taxa selected from the groupconsisting of genera Akkermansia, Anaerofilum, Bacteroides, Blautia,Bifidobacterium, Butyrivibrio, Clostridium, Coprococcus, Dialister,Dorea, Fusobacterium, Eubacterium, Faecalibacterium, Lachnospira,Lactobacillus, Phascolarctobacterium, Peptococcus, Peptostreptococcus,Prevotella, Roseburia, Ruminococcus, and Streptococcus and of thespecies Akkermansia municiphilia, Christensenella minuta, Clostridiumcoccoides, Clostridium leptum, Clostridium scindens, Dialister invisus,Eubacterium rectal, Eubacterium eligens, Faecalibacterium prausnitzii,Streptococcus salivarius, and Streptococcus thermophilus thought to beassociated with gastrointestinal health may be modulated by the glycantherapeutics described herein.

In some embodiments, the glycan therapeutics modulate (e.g.substantially increase or substantially decrease) the growth (and thetotal number) of (or substantially increase or substantially decreasethe relative representation in the total gastrointestinal community) ofone or more of (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more than 50) the genus,species, or phylogenetic clade listed in Table 1. Table 1 provides agenus level list of microbial constituents of the GI tract.

In some embodiments, the glycan therapeutics substantially increase thegrowth, e.g. the total number or the relative representation in thetotal gastrointestinal community, the community of the large intestineor the community of the small intestine of one or more of (e.g. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, or more than 50) of the OTU, genus, species, orphylogenetic clade listed in Table 1, 3, and 4.

In some embodiments, the glycan therapeutics substantially decrease thegrowth, e.g. the total number or the relative representation in thetotal gastrointestinal community, the community of the large intestineor the community of the small intestine of one or more of (e.g. 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 50, or more than 50) of the OTU, genus, species, orphylogenetic clade listed in Table 1, 3, and 4.

In some embodiments, the glycan therapeutics substantially increase anddecrease the growth, e.g. the total number or the relativerepresentation in the total gastrointestinal community, the community ofthe large intestine or the community of the small intestine of one ormore of (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, or more than 50) of the OTU, genus,species, or phylogenetic clade listed in Table 1, 3, and 4.

In some embodiments, provided herein are glycan therapeutics that aresubstrates only for a selected group bacteria that are capable ofutilizing the glycan therapeutic as a food source. The breakdown of theglycan therapeutic then exerts beneficial effects on the health of thehost. The beneficial health effects are due to a selective stimulationof the growth and/or biological activity of a selected number ofmicrobial genera, species, or strains in the gastrointestinal microbiotathat are capable of utilizing the glycan therapeutic as a food sourceand confer health benefits to the host. The effects of the glycantherapeutic, in certain embodiments, are due to selective stimulation ofthe growth of the beneficial bacteria in the GI tract. Such increasesand decreases in the abundance of certain taxa may be sufficient to“normalize” the resident microbiota, e.g. to reinstate a healthy stateor equilibrium. Increase or decrease is with respect to the ratiopresent in the human subject prior to ingestion of the pharmaceuticalglycan therapeutic composition, or to a control group not taking thepharmaceutical glycan therapeutic composition. The prebiotic index (PI)can be used as a proxy for effects of the glycan therapeutics describedherein. PI relates to the sum of: (Bifidobacteria/totalbacteria)+(Lactobacilli/total bacteria)−(Bacteroides/totalbacteria)−(Clostridia/total bacteria), (see Palframan et al, 2003, LettAppl Microbiol 37:281-284). In some embodiments, the ratio ofEubacterium rectale/total bacteria may also be considered. Eubacteriumrectale produces butyrate which is advantageous for the gut barrier inadults.

For example, the stimulation of growth of certain bacterial taxa mayreduce the pH of the colon, increase the production of short chain fattyacids, prevent the proliferation and adhesion of pathogenicmicroorganisms (barrier effect), increase the metabolism of potentiallycarcinogenic aminated compounds, and/or increase the production ofvitamins.

In some embodiments, provided herein are glycan therapeutics that can bedigested by the microbiota (e.g. by carbohydrate fermentation) withoutcertain side effects or with a substantial reduction of symptoms offermentation, such as increased gas formation that may cause flatulence,discomfort, and/or bloating.

In certain embodiments, the ratio of certain bacterial taxa or theirrelative abundance may be shifted. Such shifts may be measured withrespect to the ratio present in the subject prior to administration ofthe pharmaceutical glycan therapeutic composition, or to a control groupnot taking the pharmaceutical glycan therapeutic composition.

In some embodiments, the glycan therapeutic is a selective substrate forone or a limited number of potentially beneficial bacterial taxa thatreside in the GI tract, stimulating their growth and/or metabolicactivity. In some embodiments, the glycan therapeutic is capable ofaltering the composition of gastrointestinal microbiota to a compositionhigher or lower in specific bacterial taxa. In some embodiments, theglycan therapeutic selectively stimulates the growth and/or selectiveactivity of gastrointestinal bacterial taxa associated with health andwell-being.

Methods are provided that comprise administering to a subject in needthereof a pharmaceutical glycan therapeutic composition in an amounteffective to modulate microbial diversity. In some embodiments,administration of the glycan therapeutic modulates (e.g. increases ordecreases) microbial diversity in the GI tract (or specifically in thelarge intestine or the small intestine) of a human subject. Thediversity may increase or decrease when an effective amount of theglycan therapeutic is administered.

In some embodiments, the glycan therapeutic increases diversity. In someembodiments, the glycan therapeutic decreases diversity. Exemplaryglycan therapeutics that modulate microbial diversity include glu100,ara100, xyl100, glu50gal50, and glu33gal33fuc33.

In some embodiments, a dysbiosis has shifted the microbiota and hasincreased or decreased the microbial diversity such that a disturbedstate is reached. Administration of the glycan therapeutic can modulatethe microbial diversity, thereby treating the dysbiosis. In someembodiments, the microbial diversity is decreased and the abundance ofone or more, two or more, three or more, or four or more bacterial taxais increased, including Akkermansia, Blautia, Bacteroides,Bifidobacterium Lactobacillus, and Parabacteroides.

Microbial diversity can be measured by any suitable method known in theart, including analysis of 16S rDNA sequences described herein.Diversity can be expressed, e.g. using the Shannon Diversity index(Shannon entropy), number of observed OTUs, Chao1 index, etc. In someembodiments, the glycan therapeutics modulate (e.g. increase ordecrease) diversity within a microbial community, e.g. that of the GItract, which may be expressed using Shannon entropy as a measure.

In some embodiments, the glycan therapeutics increase microbialdiversity and associated Shannon entropy by 0.0001%, 0.0005%, 0.001%,0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 50%, 100%, 500%, 1000%,5000%, or 10000%. In some embodiments, the glycan therapeutics increasemicrobial diversity and associated Shannon entropy by (log) 1-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, or more. In some embodiments, the glycan therapeutics decreasemicrobial diversity and associated Shannon entropy by 0.0001%, 0.0005%,0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, or 99% or more. In some embodiments, the glycantherapeutics decrease microbial diversity and associated Shannon entropyby (log) 1-fold, 2-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold,70-fold, 80-fold, 90-fold, 100-fold, or more.

In some embodiments, the glycan therapeutics increase microbialdiversity and associated Shannon entropy by at least 1%, 2%, 3%, 4%, 5%,10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, or by at least 50%.

In some embodiments, the glycan therapeutics increase microbialdiversity and associated Shannon entropy by at least (log) 0.2-fold,0.3-fold, 0.4-fold, 0.5-fold, 0.8-fold, 1-fold, 1.2-fold, 1.5-fold,1.8-fold, or at least 2-fold.

In some embodiments, the glycan therapeutics decrease microbialdiversity and associated Shannon entropy by at least 1%, 2%, 3%, 4%, 5%,10%, 15% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or by atleast 75%.

In some embodiments, the glycan therapeutics decrease microbialdiversity and associated Shannon entropy by at least (log) 0.2-fold,0.3-fold, 0.4-fold, 0.5-fold, 0.8-fold, 1-fold, 1.2-fold, 1.5-fold,1.8-fold, 2-fold, 3-fold, 4-fold, or at least 5-fold.

Some methods described herein include the administration of glycantherapeutics to modulate the host's immune functions and intestinalepithelial cell functions. The glycan therapeutics may upregulate theimmune function, e.g. to improve the ability of the host to fightinfections, while downregulation of immune function may prevent theonset of allergy or intestinal inflammation. Modulated beneficialbacteria may stimulate intestinal epithelial cell responses, includingrestitution of damaged epithelial barrier, production of antibacterialsubstances and cell-protective proteins, and blocking ofcytokine-induced intestinal epithelial cell apoptosis.

Bacteria can elicit both pro- and anti-inflammatory responses from host(mammalian) cells, and different bacterial species can elicit differenthost responses. In one embodiment, glycan therapeutics are used to alterthe bacterial population to elicit a desired host response. The hostresponse may be modulated via direct interactions with the bacterialpopulation or via indirect interactions via secreted or shed bacterialproducts (e.g., short-chain fatty acids). Glycan therapeutics may alterthe bacterial population such that the bacterial population, upon eitherdirect or indirect interaction with host cells, stimulates theproduction of antimicrobial peptides (AMPs), or modulates (i.e.,increases or decreases the production of) inflammatory andimmunomodulatory cytokines including interleukin-1α (IL-1α), IL-1β,IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-17A, IL-17F, IL-22,IL-23, tumor necrosis factor (TNF), chemokine (C-C motif) ligand 5(CCL5, also known as RANTES), transforming growth factor beta (TGF-β),interferon gamma (IFN-γ), or modulates other innate or adaptive immuneresponses.

In some embodiments, the inflammatory state of the GI tract is modulatedby oral administration of a glycan therapeutic. In some embodiments,bacterial fermentation of glycan therapeutics in the gut producesshort-chain fatty acids (SCFAs). SCFAs produced by the gut microbiotaserve as energy sources for colonic epithelial cells and are thought tocontribute to the maintenance of gut barrier function, which in turnlimits plasma endotoxin levels and prevents systemic inflammation (Caniet al., Changes in gut microbiota control inflammation in obese micethrough a mechanism involving GLP-2-driven improvement of gutpermeability, Gut, 2009, 58:1091). In addition, SCFAs promote thegeneration of regulatory T (Treg) cells, and are thought to play a rolein limiting inflammatory responses (Arpaia et al., Metabolites producedby commensal bacteria promote peripheral regulatory T-cell generation,Nature, 2013, 504:451). In some embodiments, glycan therapeutics areadministered to induce systemic effects, e.g. of SCFAs and othermicrobially produced immunomodulatory molecules or metabolites tomodulate the inflammatory state of distal sites.

The glycan therapeutics when administered to a subject in an effectiveamount may modulate the production of one or more microbial metabolites,such as those listed in Table 2. In some embodiments, glycantherapeutics when administered to a subject in an effective amount maymodulate (e.g. increase or decrease) one or more of the followingmicrobial metabolites: formic acid, acetic acid, propionic acid, butryicacid, isobutyric acid, valeric acid, isovaleric acid, ascorbic acid,lactic acid, tryptophan, serotonin, and/or indole. In some embodiments,glycan therapeutics when administered to a subject in an effectiveamount may modulate (e.g. increase or decrease) one or more of thefollowing microbial metabolites: succinic acid, trimethylamine (TMA),TMAO (trimethylamine N-oxide), deoxy cholic acid, ethyphenyl sulfate,acetylaldehyde, hydrogen peroxide, and/or butanedione. In someembodiments, a substantial increase or decrease in a metabolite may bedetected. In some embodiments, the glycan therapeutic is digested by thegut microbiota (e.g. Clostridia), resulting, e.g., in the release ofshort-chain fatty acids such as butyrate, acetate, and propionate, whichmay act immunomodulatory (e.g. anti-inflammatory) and other metabolites(e.g. bile acids, and lactate) that may confer beneficial health effectson the host.

The glycan therapeutics when administered to a subject in an effectiveamount may modulate one or more host pathways. Short chain fatty acids(SCFAs) are bacterial metabolites produced in the gut by commensalbacteria including members of the families Ruminocaccaceae andLachnospiraceae (Vital M, Howe A C, Tiedje J M. 2014. Revealing thebacterial butyrate synthesis pathways by analyzing (meta)genomic data.mBio 5(2):e00889-14. doi:10.1128/mBio.00889-14). SCFAs modulate a numberof human immunological factors; for example, treatment with propionate,a SCFA, in mice or in vitro increased expression of Foxp3, a T cellregulatory factor, and IL-10, an anti-inflammatory cytokine, in colonicregulatory T cells. Additionally, exposure to SCFAs has been shown toincrease frequency and number of colonic regulatory T cells (cTregs) andCD4+ T cells in germ-free mice (Smith P M et al. 2013. The microbialmetabolites, short chain fatty acids, regulate colonic Treg cellhomeostasis. Science; 341(6145). SCFAs promote gut barrier function byaffecting mucin production and gastrointestinal peptide LL-37, and SCFAsadditionally modulate inflammation by suppressing NF-kB and theproduction of inflammatory cytokines such as IL-6 and TNF-α (Kim C H etal. 2014. Gut Microbiota-Derived Short-Chain Fatty Acids, T Cells, andInflammation. Immune Network 14(6):277-288). In some embodiments, glycantherapeutics when administered in an effective amount modulate bacterialspecies that produce SCFAs, such as, e.g., those of the Ruminocacceaefamily and/or Lachnospiraceae family. In some embodiments, the glycantherapeutics modulate host immunity and inflammation. For example, inthe in vitro assay of Example 8, growth of ROB.74, a member of theRuminocacceae family, was supported by 13 out of 15 glycans, and growthof CSC.32 and CNE.31, members of the Lachnospiraceae family, weresupported by 6 and 7 out of 15 glycans, respectively.

In some embodiments, method of modulating a functional pathway of themicrobiota of the gastrointestinal tract are provided. The methodsinclude administering to the human subject a pharmaceutical compositioncomprising a glycan therapeutic preparation in an amount effective tomodulate the functional pathway. In some embodiments, the functionalpathway modulates the production of anti-microbial agent, a secondarybile acid, a short-chain fatty acid, a siderophore or a metabolitelisted in Table 2 by the microbiota. In some embodiments, the shortchain fatty acid is produced by one or more bacterial member of theRuminocaccaceae and/or Lachnospiraceae family. In some embodiments, thesubject is obese.

In some embodiments, the pharmaceutical glycan therapeutic compositionscomprise one or more polyphenols. The glycan therapeutic preparation andthe one or more polyphenols in the pharmaceutical composition can haveadditive or synergistic effects.

In some embodiments, the polyphenols are capable of modulating one ormore bacterial constituents in the GI tract (e.g. for polyphenols fromcranberry extract: Anhê F F et al. et al. “A polyphenol-rich cranberryextract protects from diet-induced obesity, insulin resistance andintestinal inflammation in association with increased Akkermansia spp.population in the gut microbiota of mice.” Gut. 2014; for blueberryextract: Guglielmetti S et al. “Differential modulation of humanintestinal bifidobacterium populations after consumption of a wildblueberry (Vaccinium angustifolium) drink. J Agric Food Chem. 2013;61(34):8134-40; Lacombe A et al. “Phytochemicals in lowbush wildblueberry inactivate Escherichia coli O157:H7 by damaging its cellmembrane.” Foodborne Pathog Dis. 2013; 10(11): 944-50; for grapeextracts: Choy Y Y et al. “Phenolic metabolites and substantialmicrobiome changes in pig feces by ingesting grape seedproanthocyanidins.” Food Funct. 2014; 5(9):2298-308; Roopchand D E etal. “Dietary polyphenols promote growth of the gut bacterium Akkermansiamuciniphila and attenuate high fat diet-induced metabolic syndrome.”Diabetes. 2015; for peach and plum extract: Noratto G D et al.“Carbohydrate-free peach (Prunus persica) and plum (Prunus domestica)juice affects fecal microbial ecology in an obese animal model.” PLoSOne. 2014; 9(7):e101723; for red wine and black tea: Kemperman R A,Gross G, Mondot S, et al. Impact of polyphenols from black tea and redwine/grape juice on a gut model microbiome. Food Res Int. 2013,53(2):659-69; for soy (legume): Rafii F “The role of colonic bacteria inthe metabolism of the natural isoflavone daidzin to equol.” Metabolites.2015 Jan. 14:56-73).

In some embodiments, the pharmaceutical composition comprising theglycan therapeutic preparation and the polyphenol preparation modulates(e.g. increases or decreases) the growth of one or more bacterial taxa,such as bacteria of the phylum Verrucomicrobia, e.g., those of the genusAkkermansia. In some embodiments, the pharmaceutical compositioncomprising the glycan therapeutic preparation and the polyphenolpreparation increases the abundance of bacteria of the phylumVerrucomicrobia, including the genus Akkermansia

In some embodiments, polyphenols in the compositions have antioxidantfunctions. In some embodiments, polyphenols in the compositions haveanti-bacterial functions. In some embodiments, the antioxidant and/oranti-bacterial function of the polyphenols in the composition modulatesthe abundance of one or more bacteria residing in the GI tract.

In some embodiments, the pharmaceutical glycan therapeutic compositioncomprises polyphenols that act as antimicrobials, e.g., by inhibitingthe growth of subsets of species, such as, e.g. pathogens orpathobionts. (Puupponen-Pimiä R et al. “Antimicrobial properties ofphenolic compounds from berries.” 2001. Journal of Applied Microbiology90: 494-507; Puupponen-Pimiä R et al. “Berry phenolics selectivelyinhibit the growth of intestinal pathogens.” 2005. Journal of AppliedMicrobiology 98: 991-1000).

In some embodiments, polyphenols in the composition are a selectivesubstrate for one or more bacterial taxa that reside in the GI tract,(e.g., Selma M V et al. “Interaction between Phenolics and GutMicrobiota: Role in Human Health.” 2009. Journal of Agricultural andFood Chemistry 57: 6485-6501; Déprez S et al. “PolymericProanthocyanidins Are Catabolized by Human Colonic Microflora intoLow-Molecular-Weight Phenolic Acids.” 2000. The Journal of Nutrition131: 2733-2738; Tzounis X et al. “Flavanol monomer-induced changes tothe human faecal microflora.” 2007. The British Journal of Nutrition 99:782-792; Kutschera M et al. “Isolation of catechin-converting humanintestinal bacteria.” 2011. Journal of Applied Microbiology 111:165-175; Schneider H et al. “Anaerobic transformation ofquercetin-3-glucoside by bacteria from the human intestinal tract.”1999. Archives of Microbiology 171: 81-91; Hein E M et al.“Deconjugation and Degradation of Flavonol Glycosides by Pig CecalMicrobiota Characterized by Fluorescence in Situ Hybridization (FISH).”2008. Journal of Agricultural and Food Chemistry 56: 2281-2290).

Methods of Screening a Plurality of Glycan Therapeutic Preparations

In order to characterize the effects of the glycan therapeutics,provided is an in vitro microplate-based screening system thatdemonstrates the efficacy of the glycan therapeutic preparation,including the ability to inhibit (or antagonize/suppress) the growth ofcertain microbial constituents and the ability to promote (or increase)the growth of other microbial constituents. These methods provide novelglycan therapeutic preparations that are able to improve the health ofthe gastrointestinal microbiome and/or promote health of the subject. Insome embodiments, the screening methods include: i) providing aplurality of preparations of glycan therapeutics, ii) subjecting thepreparation to one or more screen, iii) selecting a preparation ofglycan therapeutics based on the screens, and optionally iv) isolatingthe selected preparation of glycan therapeutics. A suitable singlestrain screening method is described in the Examples. Other suitablescreens are known to one of ordinary skill and any necessaryexperimental parameters may be adjusted with only routineexperimentation.

In some embodiments, glycan therapeutics promote the growth of bacterialstrains that are able to significantly reduce the rate of pathogengrowth and/or capable of partially or fully restoring a bacterialcommunity that is associated with a healthy GI tract.

In some embodiments, glycan therapeutics are provided that promote thegrowth of beneficial bacteria. Exemplary glycans are listed in Table 8.In some embodiments, commensal growth-promoting glycan therapeuticinclude gal100, glu100, xyl100, ara100, ara50gal50, ara50xyl50,gal75xyl25, glu50gal50, man62glu38, glu33gal33fuc33 and man52glu29gal19.

In some embodiments, glycan therapeutics are provided that do notpromote the growth of pathogenic bacteria but promote the growth ofbeneficial bacteria. Exemplary glycans are listed in Table 9. In someembodiments, commensal-selective glycan therapeutic (e.g. a glycantherapeutic that preferentially promotes the growth of commensalbacteria over the growth of pathogenic bacteria) include gal100, glu100,xyl100, ara50gal50, and ara50xyl50.

In some embodiments, methods are provided that include selecting aglycan therapeutic for further processing (e.g. formulating it into apharmaceutical composition) or further testing (e.g. analyzingadditional characteristics) using a single strain screen. For example,the glycan therapeutic may be tested for promoting growth in mediasupplemented with the preparation of commensal strains selected from thegroup consisting of Bacteroides caccae ATCC 43185, Prevotella copri DSM18205, Bacteroides thetaiotaomicron ATCC 29741, Bacteroidescellulosilyticus DSM 14838, Clostridium scindens ATCC 35704,Ruminococcus obeum ATCC 29714, Clostridium nexile ATCC 27757, andParabacteroides distasonis ATCC 8503. Alternatively or in addition, theglycan therapeutic may be tested for promoting growth in mediasupplemented with the preparation of pathogenic strains selected fromthe group consisting of Clostridium difficile ATCC BAA-1382, Clostridiumdifficile ATCC 43255, Enterococcus faecium ATCC 700221, and Salmonellaenterica ATCC 27869. A glycan therapeutic may be selected for furtherprocessing or testing if one or both of the following criteria are met:i) the glycan therapeutic promotes the growth of at least 4, 5, or atleast 6 commensal strains, and ii) the glycan therapeutic promotes thegrowth of no more than 3, 2, 1 or no more than 0 pathogenic strains.

The effect of the glycan therapeutics on bacterial growth can also betested in other in vitro assays and using laboratory animal models. Thebacteria can be collected from samples taken from the niche of interest(e.g. a stool sample containing feces) and propagated by methods knownin the art. Competitive in vitro growth assays may then be performedusing conditions that are suitable for growth of bacteria from the nicheof interest, e.g. conditions that may mimic the natural environment ofthe niche, e.g. the GI tract or a subset thereof, such as the large andsmall intestine. Such conditions include, but are not limited toaerobic, anaerobic, low/high/neutral pH, physiological temperature (e.g.human body temperature), etc.

In some embodiments, in vivo assays are performed to detect the effectof the glycan therapeutic on bacterial growth in the GI tract. In orderto determine whether the glycan therapeutic preparation modulates themicrobial populations in the GI tract of a subject, a laboratory animalmodel, such as a mouse model of human disease, can be used. Themicrobiota of the mice can be evaluated and characterized. Qualitativeassessments can be accomplished using 16S rRNA profiling of themicrobial community in the GI tract of normal mice. It can also beaccomplished by full genome sequencing, whole genome shotgun sequencing(WGS), or traditional microbiological techniques. Quantitativeassessments can be conducted using quantitative PCR (qPCR) or by usingtraditional microbiological techniques and counting colony formation.Optionally, the mice can receive an antibiotic treatment to mimic thecondition of a disturbed gastrointestinal microbiota in which the GImicrobiota exhibit a dysbiosis. It is known that antibiotic treatmentcan decrease the taxonomic richness, diversity, and evenness of gutcommunities, including a reduction of abundance of a significant numberof bacterial taxa. (Dethlefsen et al., The pervasive effects of anantibiotic on the human gut microbiota, as revealed by deep 16S rRNAsequencing, PLoS Biology 6(11):3280 (2008)).

Various effects of glycan therapeutics (e.g. to assess modulation ofbacterial taxa, modulation of microbial diversity, modulation ofdrug-induced symptoms, and therapeutic effects (e.g. assessing themodulation of disease-associated phenotypes)), can be assessed insuitable animal models for a certain disease, such as, e.g., aDSS-colitis mouse model (e.g. to assess diseases, disorders orconditions associated with an inflammation or drug-induced damage), adiet-induced obesity mouse model (e.g. to assess metabolic diseases,disorders or conditions) and a C. difficile infection mouse model (e.g.to assess diseases, disorders or conditions associated with an infectionor drug-induced damage), and wild-type mouse models subjected to, e.g.,drug-treatments (e.g. antibiotic regimen, cancer drug regimen, etc.) ordiet-changes, such as, e.g. zero-fiber diet, low-fiber diets, normalchow diet, high-fat diet, etc. to assess various states of themicrobiota of the gastrointestinal tract.

In some embodiments, the screening methods are carried out using asuitable laboratory animal model. For example, a preparation of glycantherapeutics may be administered to a laboratory animal and after aperiod of time a sample is taken from the laboratory animal's GI tractand analyzed for growth of bacterial taxa. The laboratory animal may, ifdesired, be contacted with pathogens or other bacteria to facilitatecolonization of the animal prior to or concurrent with administration ofthe glycan therapeutic. In some embodiments, a preparation of glycantherapeutics is selected that is capable of modulating (e.g. increasingor decreasing) the growth of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,or at least 20 bacterial taxa in the laboratory animal.

In one embodiment, the animal model is a C. difficile mouse model andthe glycan therapeutic is capable of modulating one or more ofPrevotella, Akkermansia, Bacteroides, Clostridium (Erysipelotrichaceae),Clostridium (Clostridiaceae), Bifidobacterium, Aggregatibacter,Clostridium (Peptostreptococcaveae), Parabacteroides, Lactobacillus, andEnterococcus. In one embodiment, the animal model is a zero-fiber andnormal chow wild-type mouse model and the glycan therapeutic is capableof modulating Akkermansia and Blautia. In some embodiments, thetherapeutic glycan is xyl100, glu100, glu33gal33fuc33, glu50gal50, orara100.

In some embodiments, the screen is an in vitro assay in which one ormore bacterial taxa are grown in a growth medium and the growth ismonitored in the presence of the glycan therapeutics and compared togrowth in the absence of the glycan therapeutics. Any practical numberof bacterial taxa may be grown in the medium, such as, e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 taxa. In some embodiments, apreparation of glycan therapeutics is selected that modulates (e.g.increases or decreases) the growth of at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, or at least 20 bacterial taxa. In one embodiment, the screenis a single strain assay and the glycan therapeutic is selected fromthose listed in Table 8 modifying at least 5, 6, 7, or 8 strains ofTable 8.

In some embodiments, the growth of one or more bacterium is increased byat least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 110%, 120%,130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%,450%, 500%, 600%, 700%, 800%, 900%, or by at least 1000% after 1 hour, 6hours, 12 hours, 18 hours, 24 hours, 48 hours or 72 hours of contacting.

In other embodiments, the growth of one or more bacterium is decreasedby at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or by at least 99.9% after 1 hour, 6 hours, 12 hours, 18hours, 24 hours, 48 hours or 72 hours of contacting.

In some embodiments, the glycan therapeutic also modulates theconcentration of one or more microbial metabolite selected from thegroup consisting of the metabolites listed in Table 2. In someembodiments, the metabolite concentration is increased by at least 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 110%, 120%, 130%, 140%,150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450%, 500%,600%, 700%, 800%, 900%, or by at least 1000% after 1 hour, 6 hours, 12hours, 18 hours, 24 hours, 48 hours or 72 hours of contacting. In otherembodiments, the metabolite concentration is decreased by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, orby at least 99.9% after 1 hour, 6 hours, 12 hours, 18 hours, 24 hours,48 hours or 72 hours of contacting.

Digestibility is a parameter that can be ascertained for the glycantherapeutics described herein. In some embodiments, glycan therapeuticsdisclosed herein are screened to assess their digestibility.Digestibility of glycan therapeutics can be assessed by any suitablemethod known in the art. In some embodiments, digestibility is assessedby a physiologically relevant in vitro digestion reaction. Samples atdifferent stages of the digestion can be analyzed by standard glycantechniques known in the art and described herein. By monitoring theamount of intact glycan therapeutics observed over time, the half-lifeof digestion can be calculated. Suitable assays can be used to assesscomparative digestibility (e.g., against a benchmark glycan) or toassess absolute digestibility.

Digestibility of a glycan therapeutic is a function of the number orrepresentation of hydrolysable glycosidic bonds in the glycan species ofthe preparation. Enzymes that are capable of hydrolyzing glycosidicbonds usually are specific to a particular bond, stereochemistry, and asubunit composition. Certain types of hydrolyzable bonds, e.g., alpha1,4; alpha 1,6, alpha 1,2; and alpha 1,6 glycosidic linkages arerecognized by specific microbial enzymes (e.g. alpha-glucosidase,cyclomaltodextrinase, neopullunanase, glucanotransferase,trehalohydrolase, and the like) and are not substrates for mammalianenzymes. Digestibility of glycans depends on many factors, including,e.g., the degree of polymerization, the degree of branching, the type ofglycosidic linkages, position of the linkages, anomeric configuration(e.g. L- or D-configuration, alpha/beta configuration) of the glycanunit(s) (e.g. monosaccharide), and the glycan unit composition. Forexample, furanosides are generally more susceptible to hydrolysis thanpyranosides. Deoxy sugars are generally more acid labile than non-deoxysugars. Uronic acids are generally less susceptible to hydrolysis thannon-uronic monosaccharides. Branching protects against digestion byhuman enzymes, and it is generally observed that the larger themolecule, the lesser the fermentation speed (digestibility) in thecolon. These characteristics generally promote indigestibility by humanglycosidases and can promote selective fermentation or digestion by themicrobiota.

In some embodiments, pharmaceutical glycan therapeutic compositions thatare administered orally and that reach the gut comprise a mixture of aplurality of glycan species with a desired degree of digestibility inthe gut (or specific regions of the gut) of the host. In someembodiments, the glycan therapeutic is non-digestible to mammalianenzymes and can only be hydrolyzed by microbial enzymes. In someembodiments, the glycan therapeutic cannot be metabolized by a human andis only metabolizable (or fermentable) by the human's microbiota.

Different microbial taxa have different hydrolyzing enzymes. In someembodiments, the glycan therapeutic is fermentable in an in vitro singlestrain digestibility assay by one, two, three, four, five or morecommensal bacterial species, e.g. Bacteroides caccae ATCC 43185,Prevotella copri DSM 18205, Bacteroides thetaiotamicron ATCC 29741,Bacteroides cellulosilyticus DSM 14838, Clostridium scindens ATCC 35704,Ruminococcus obeum ATCC 29714, Clostridium nexile ATCC 27757, andParabacteroides distasonis ATCC 8503. In some embodiments, the glycantherapeutic is non-fermentable in an in vitro single straindigestibility assay by one, two, three, four, five or more pathogenicspecies, e.g., of Clostridium difficile ATCC BAA-1382, Clostridiumdifficile ATCC 43255, Enterococcus faecium ATCC 700221, and Salmonellaenterica ATCC 27869. In some embodiments, the glycan therapeutic isnon-fermentable by a specific bacterial taxa in a single strain in vitrodigestibility assay (e.g. at least 70%, 80%, 90%, 95% or 98% of theglycan preparation is non-fermentable) but is fermentable by the taxa invivo in a suitable bacterial niche of the host (e.g. the GI tract or aspecific region thereof, such as the colon or intestine). In someembodiments, the bacterial taxa include Akkermansia, Bacteroides,Bifidobacterium, Lactobacillus, and Parabacteroides. Fermentability canbe measured, e.g., by monitoring growth of the bacterial taxa in vitroor in vivo.

In some embodiments, hydrolysis of glycosidic bonds are catalyzed by anenzyme and the rate of catalysis can be measured by any suitable meansknown in the art and the rate can be compared to that of another enzyme.A high rate of hydrolysis, transfer of glycan units, and/or modificationof glycan units may suggest that the bond is a suitable substrate of theenzyme. Ease of hydrolysis can be expressed by a high rate of catalyticreaction. Other bonds are incompatible with an enzyme or a set ofenzymes and they are difficult to hydrolyze. In some embodiments, thedigestibility (expressed as half-life) is 30 minutes or less, 20 minutesor less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4minutes or less, 3 minutes or less, 2 minutes or less or 1 minute orless. In some embodiments, the digestibility (expressed as half-life) is30 minutes or more, 45 minutes or more, 1 hour or more, 2 hours or more,3 hours or more, 4 hours or more, 5 hours or more, or 10 hours or more.In some embodiments, the preparation of glycan therapeutics comprisesless than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%,20%, 30%, 40%, or less than 50% bonds that are hydrolyzable by amammalian amylase enzyme. Digestibility may also be assessed by gastricdigestion half-life.

Identification of Bacterial Constituents

In some embodiments, the pharmaceutical glycan therapeutic compositionsdescribed herein are administered to a subject to increase the growth ofbeneficial bacteria and/or to decrease the growth of pathogens in the GItract. In some embodiments, the microbial community is shifted by theglycan therapeutic toward that of a healthy state. The microbial changesoccurring in the GI tract can be analyzed using any number of methodsknown in the art and described herein.

As one quantitative method for determining whether a glycan therapeuticpreparation results in a shift of the population of bacteria in the GItract, quantitative PCR (qPCR) can be performed. Genomic DNA can beextracted from samples using commercially-available kits, such as the MoBio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo Bio Laboratories,Carlsbad, Calif.), the Mo Bio Powersoil® DNA Isolation Kit (Mo BioLaboratories, Carlsbad, Calif.), or the QIAamp DNA Stool Mini Kit(QIAGEN, Valencia, Calif.) according to the manufacturer's instructions,or by other standard methods known to those skilled in the art.

In some embodiments, qPCR can be conducted using HotMasterMix (5PRIME,Gaithersburg, Md.) and primers specific for certain (e.g. beneficial ordesired) bacteria and may be conducted on a MicroAmp® Fast Optical96-well Reaction Plate with Barcode (0.1 mL) (Life Technologies, GrandIsland, N.Y.) and performed on a BioRad C1000™ Thermal Cycler equippedwith a CFX96™ Real-Time System (BioRad, Hercules, Calif.), withfluorescent readings of the FAM and ROX channels. The Cq value for eachwell on the FAM channel is determined by the CFX Manager™ softwareversion 2.1. The log₁₀ (cfu/ml) of each experimental sample iscalculated by inputting a given sample's Cq value into linear regressionmodel generated from the standard curve comparing the Cq values of thestandard curve wells to the known log₁₀ (cfu/ml) of those samples. Theskilled artisan may employ alternative qPCR modes.

In some embodiments, the microbial constituents are identified bycharacterizing the DNA sequence of microbial 16S small subunit ribosomalRNA gene (16S rRNA gene). 16S rRNA gene is approximately 1,500nucleotides in length, and in general is highly conserved acrossorganisms, but contain specific variable and hypervariable regions(V1-V9) that harbor sufficient nucleotide diversity to differentiatespecies- and strain-level taxa of most organisms. These regions inbacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682,822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465 respectively usingnumbering based on the E. coli system of nomenclature. (See, e.g.,Brosius et al., Complete nucleotide sequence of a 16S ribosomal RNA genefrom Escherichia coli, PNAS 75(10):4801-4805 (1978)).

Composition of a microbial community can be deduced by sequencing full16S rRNA gene, or at least one of the V1, V2, V3, V4, V5, V6, V7, V8,and V9 regions of this gene or by sequencing of any combination ofvariable regions from this gene (e.g. V1-3 or V3-5). In one embodiment,the V1, V2, and V3 regions are used to characterize a microbiota. Inanother embodiment, the V3, V4, and V5 regions are used to characterizea microbiota. In another embodiment, the V4 region is used tocharacterize a microbiota.

Sequences that are at least 97% identical to each other are grouped intoOperational Taxonomic Units (OTUs). OTUs that contain sequences with 97%similarity correspond to approximately species level taxa. At least onerepresentative sequence from each OTU is chosen, and is used to obtain ataxonomic assignment for an OTU by comparison to a reference database ofhighly curated 16S rRNA gene sequences (such as Greengenes or SILVAdatabases). Relationship between OTUs in a microbial community could bededuces by constructing a phylogenetic tree from representativesequences from each OTU.

Using known techniques, in order to determine the full 16S sequence orthe sequence of any variable region of the 16S sequence, genomic DNA isextracted from a bacterial sample, the 16S rRNA (full region or specificvariable regions) amplified using polymerase chain reaction (PCR), thePCR products are cleaned, and nucleotide sequences delineated todetermine the genetic composition of 16S rRNA gene or a variable regionof the gene. If full 16S sequencing is performed, the sequencing methodused may be, but is not limited to, Sanger sequencing. If one or morevariable regions is used, such as the V4 region, the sequencing can be,but is not limited to being performed using the Sanger method or using anext-generation sequencing method, such as an Illumina method. Primersdesigned to anneal to conserved regions of 16S rRNA genes (e.g., the515F and 805R primers for amplification of the V4 region) could containunique barcode sequences to allow characterizing multiple microbialcommunities simultaneously.

As another method to identify microbial composition is characterizationof nucleotide markers or genes, in particular highly conserved genes(e.g., “house-keeping” genes), or a combination thereof, or whole genomeshotgun sequence (WGS). Using defined methods, DNA extracted from abacterial sample will have specific genomic regions amplified using PCRand sequenced to determine the nucleotide sequence of the amplifiedproducts. In the WGS method, extracted DNA will be fragmented intopieces of various lengths (from 300 to about 40,000 nucleotides) anddirectly sequenced without amplification. Sequence data can be generatedusing any sequencing technology including, but not limited to Sanger,Illumina, 454 Life Sciences, Ion Torrent, ABI, Pacific Biosciences,and/or Oxford Nanopore.

In addition to the 16S rRNA gene, a selected set of genes that are knownto be marker genes for a given species or taxonomic group is analyzed toassess the composition of a microbial community. These genes arealternatively assayed using a PCR-based screening strategy. For example,various strains of pathogenic Escherichia coli are distinguished usinggenes that encode heat-labile (LTI, LTIIa, and LTIIb) and heat-stable(STI and STII) toxins, verotoxin types 1, 2, and 2e (VT1, VT2, and VT2e,respectively), cytotoxic necrotizing factors (CNF1 and CNF2), attachingand effacing mechanisms (eaeA), enteroaggregative mechanisms (Eagg), andenteroinvasive mechanisms (Einv). The optimal genes to utilize todetermine the taxonomic composition of a microbial community by use ofmarker genes are familiar to one with ordinary skill in the art ofsequence based taxonomic identification.

Sequencing libraries for microbial whole-genome sequencing (WGS) may beprepared from bacterial genomic DNA. For genomic DNA that has beenisolated from a human or laboratory animal sample, the DNA mayoptionally enriched for bacterial DNA using commercially available kits,for example, the NEBNext Microbiome DNA Enrichment Kit (New EnglandBiolabs, Ipswich, Mass.) or other enrichment kit. Sequencing librariesmay be prepared from the genomic DNA using commercially available kitsas well, such as the Nextera Mate-Pair Sample Preparation Kit, TruSeqDNA PCR-Free or TruSeq Nano DNA, or the Nextera XT Sample PreparationKit (Illumina, San Diego, Calif.) according to the manufacturer'sinstructions. Alternatively, libraries can be prepared using other kitscompatible with the Illumina sequencing platform, such as the NEBNextDNA Library Construction Kit (New England Biolabs, Ipswich, Mass.).Libraries may then be sequenced using standard sequencing technologyincluding, but not limited to, a MiSeq, HiSeq or NextSeq sequencer(Illumina, San Diego, Calif.). Alternatively, a whole-genome shotgunfragment library prepared using standard methods in the art. Forexample, the shotgun fragment library could be constructed using the GSFLX Titanium Rapid Library Preparation Kit (454 Life Sciences, Branford,Conn.), amplified using a GS FLX Titanium emPCR Kit (454 Life Sciences,Branford, Conn.), and sequenced following standard 454 pyrosequencingprotocols on a 454 sequencer (454 Life Sciences, Branford, Conn.).Bacterial RNA may be isolated from microbial cultures or samples thatcontain bacteria by commercially available kits, such as the RiboPureBacterial RNA Purification Kit (Life Technologies, Carlsbad, Calif.).Another method for isolation of bacterial RNA may involve enrichment ofmRNA in purified samples of bacterial RNA through remove of tRNA.Alternatively, RNA may be converted to cDNA, which used to generatesequencing libraries using standard methods such as the Nextera XTSample Preparation Kit (Illumina, San Diego, Calif.).

Nucleic acid sequences are analyzed to define taxonomic assignmentsusing sequence similarity and phylogenetic placement methods or acombination of the two strategies. A similar approach is used toannotate protein names, protein function, transcription factor names,and any other classification schema for nucleic acid sequences. Sequencesimilarity based methods include BLAST, BLASTx, tBLASTn, tBLASTx,RDP-classifier, DNAclust, RapSearch2, DIAMOND, USEARCH, and variousimplementations of these algorithms such as QIIME or Mothur. Thesemethods map a sequence read to a reference database and select the bestmatch. Common databases include KEGG, MetaCyc, NCBI non-redundantdatabase, Greengenes, RDP, and Silva for taxonomic assignments. Forfunctional assignments, reads are mapped to various functional databasessuch as COG, KEGG, BioCyc, MetaCyc, and the Carbohydrate-Active Enzymes(CAZy) database. Microbial clades are assigned using software includingMetaPhlAn.

Proteomic Analysis of Microbial Populations

Preparations of glycan therapeutics may be selected based on theirability to increase the expression of microbial proteins associated withhealthy states or to decrease the expression of microbial proteinsassociated with diseased states. Proteomic analysis of microbialpopulations can be performed following protocols known to one skilled inthe art (e.g., Cordwell, Exploring and exploiting bacterial proteomes,Methods in Molecular Biology, 2004, 266:115). To identify differentiallyexpressed proteins (for example, to identify changes in proteinexpression upon treatment of microbial populations with glycantherapeutics), proteomic analysis can be performed as described, e.g.,in Juste et al. (Bacterial protein signals are associated with Crohn'sdisease, Gut, 2014, 63:1566). For example, the protein is isolated fromthe microbial lysates of two samples (for example, an untreatedmicrobial population and a population that has been treated with glycantherapeutics). Each protein sample is labeled (e.g., with a fluorescentdye, e.g., Cy3 or Cy5 CyDye DIGE Fluor minimal dye, GE Healthcare) andanalyzed by two-dimensional differential gel electrophoresis (2D-DIGE).Gels are stained and protein spots identified as being significantlydifferent between the two samples are excised, digested, and analyzed byliquid chromatography-tandem mass spectrometry (LC-MS/MS).X!TandemPipeline (http://pappso.inra.fr/bioinfo/xtandempipeline/) can beused to identify differentially expressed proteins.

Preparations of glycan therapeutics may also be selected foradministration to a human subject based on their effect on the presenceof microbial fermentation products. For example, preparations of glycantherapeutics can be selected for their ability to induce or promotegrowth of bacteria that produce short chain fatty acids such aspropionate (propionic acid), acetate, and/or butyrate (butyric acid).Similarly, preparations of glycan therapeutics can be selected for theirability to induce or promote growth of bacteria that produce lacticacid, which can modulate the growth of other bacteria by producing anacidic environment. Such analysis may also be used to pair probioticbacteria with glycan therapeutics such that the glycan therapeutic is asubstrate for the production of the desired fermentation products.

The metabolites that are present in fresh or spent culture media or inbiological samples collected from humans may be determined using methodsdescribed herein. Unbiased methods that may be used to determine therelative concentration of metabolites in a sample and are known to oneskilled in the art, such as gas or liquid chromatography combined withmass spectrometry or ¹H-NMR. These measurements may be validated byrunning metabolite standards through the same analytical systems.

In the case of gas chromatography-mass spectrometry (GC-MS) orliquid-chromatography-mass spectrometry (LC-MS) analysis, polarmetabolites and fatty acids could be extracted using monophasic orbiphasic systems of organic solvents and an aqueous sample andderivatized (Fendt et al., Reductive glutamine metabolism is a functionof the α-ketoglutarate to citrate ratio in cells, Nat Commun, 2013,4:2236; Fendt et al., Metformin decreases glucose oxidation andincreases the dependency of prostate cancer cells on reductive glutaminemetabolism, Cancer Res, 2013, 73:4429; Metallo et al., Reductiveglutamine metabolism by IDH1 mediates lipogenesis under hypoxia, Nature,2011, 481:380). An exemplary protocol for derivatization of polarmetabolites involves formation of methoxime-tBDMS derivatives throughincubation of the metabolites with 2% methoxylamine hydrochloride inpyridine followed by addition ofN-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) with 1%tert-butyldimethylchlorosilane (t-BDMCS). Non-polar fractions, includingtriacylglycerides and phospholipids, may be saponified to free fattyacids and esterified to form fatty acid methyl esters, for example,either by incubation with 2% H₂SO₄ in methanol or by using Methyl-8reagent (Thermo Scientific). Derivatized samples may then be analyzed byGC-MS using standard LC-MS methods, for example, a DB-35MS column (30m×0.25 mm i.d.×0.25 μm, Agilent J&W Scientific) installed on a gaschromatograph (GC) interfaced with an mass spectrometer (MS). Massisotopomer distributions may be determined by integrating metabolite ionfragments and corrected for natural abundance using standard algorithms,such as those adapted from Fernandez et al. (Fernandez et al.,Correction of 13C mass isotopomer distributions for natural stableisotope abundance, J Mass Spectrom, 1996, 31:255). In the case of liquidchromatography-mass spectrometry (LC-MS), polar metabolites may beanalyzed using a standard benchtop LC-MS/MS equipped with a column, suchas a SeQuant ZIC-pHILIC Polymeric column (2.1×150 mm; EMD Millipore).Exemplary mobile phases used for separation could include buffers andorganic solvents adjusted to a specific pH value.

In combination or in the alternative, extracted samples may be analyzedby ¹H-nuclear magnetic resonance (¹H-NMR). Samples may be combined withisotopically enriched solvents such as D2O, optionally in the presenceof a buffered solution (e.g., Na₂HPO₄, NaH₂PO₄ in D₂O, pH 7.4). Samplesmay also be supplemented with a reference standard for calibration andchemical shift determination (e.g., 5 mM2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS-d₆, Isotec,USA)). Prior to analysis, the solution may be filtered or centrifuged toremove any sediment or precipitates, and then transferred to a suitableNMR tube or vessel for analysis (e.g., a 5 mm NMR tube). ¹H-NMR spectramay be acquired on a standard NMR spectrometer, such as an Avance II+500Bruker spectrometer (500 MHz) (Bruker, DE), equipped with a 5 mm QXI-ZC/N/P probe-head) and analyzed with spectra integration software (suchas Chenomx NMR Suite 7.1; Chenomx Inc., Edmonton, AB). (Duarte et al.,¹H-NMR protocol for exometabolome analysis of cultured mammalian cells,Methods Mol Biol, 2014:237-47). Alternatively, ¹H-NMR may be performedfollowing other published protocols known in the art (Chassaing et al.,Lack of soluble fiber drives diet-induced adiposity in mice, Am JPhysiol Gastrointest Liver Physiol, 2015; Bal et al., Comparison ofStorage Conditions for Human Vaginal Microbiome Studies, PLoS ONE,2012:e36934).

Methods of Treatment

Provided herein are methods for treating a human subject. These methods,in some embodiments, include one or both of i) identifying a humansubject having or suspected of having a dysbiosis of thegastrointestinal microbiota, and ii) administering to the human subjecta pharmaceutical composition comprising a glycan therapeutic preparationin an amount effective to treat the dysbiosis.

The pharmaceutical glycan therapeutic compositions described herein aresuitable for administration to humans in need thereof. In certainembodiments, the subject is a human that has one or more symptoms of adysbiosis of the gastrointestinal microbiota, including but not limitedto overgrowth of an undesired pathogen or one or more undesiredbacterial taxa, reduced representation of key health-associatedbacterial taxa, reduced or increased diversity of microbial speciescompared to a healthy individual, or reduced overall abundance ofbeneficial bacteria.

In some embodiments, the glycan therapeutics are beneficial in thetreatment of various diseases, disorders or conditions. Such disease,disorders or conditions may be associated with a dysbiosis of themicrobiota. Disturbances in beneficial microbiota can occur due to avariety of factors (e.g. genetic or environmental) including, but notlimited to, use of antibiotics, chemotherapeutics and otherdysbiosis-inducing drugs or treatments (e.g. radiation treatment),pathogen infection, pathobiont activity, miscalibrated caloric intake(e.g. high-fat, high-sugar), miscalibrated (non-digestible) fiber intake(e.g. low or zero fiber), host factors (e.g. host genetic alterations),and similar.

In some embodiments, the disease, disorder or condition is associatedwith a dysbiosis of the gastrointestinal microbiota. In someembodiments, by treating the dysbiosis the disease, disorder orcondition is treated.

Symptoms that may be associated with a dysbiosis of the gastrointestinalmicrobiota and/or with a gastrointestinal disease, disorder or conditioninclude, but are not limited to gas, heartburn, stomach upset, bloating,flatulence, diarrhea, abdominal pain, cramping, nausea, and vomiting.Minor digestive problems related to the GI also include occasionalbloating, diarrhea, constipation, gas, or stomach upset.

Infectious Diseases

In some embodiments, administration of the glycan therapeutic reducesinfection. In some embodiments, a subject is identified to be suitablefor treatment if the subject has or is suspected of having a disease,disorder or condition including: gastrointestinal infectious diseasesincluding Clostridium difficile infection (CDI); Vancomycin-resistantenterococci (VRE) infection, infectious colitis, and C. difficilecolitis; mycoses, such as, e.g., Candida albicans infection,Campylobacter jejuni infection, Helicobacter pylori infection; diarrhea,such as, e.g., Clostridium difficile associated diarrhea (CDAD),antibiotic-associated diarrhea (AAD), antibiotic-induced diarrhea,travellers' diarrhea (TD), pediatric diarrhea, (acute) infectiousdiarrhea, colon and liver cancers, ameboma; necrotizing enterocolitis(NEC), and small intestine bacterial overgrowth (SIBO); indigestion ornon-ulcer dyspepsia; anal fissures, perianal abscess and anal fistula;diverticulosis or diverticulitis; peptic ulcers; and gastroenteritis.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of having aClostridium difficile infection (CDI); a Vancomycin-resistantenterococci (VRE) infection, infectious colitis, or C. difficilecolitis.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingmycoses, such as, e.g., Candida albicans infection, Campylobacter jejuniinfection, or Helicobacter pylori infection.

In some embodiments, the GI tract infection is a bacterial or viralinfection, such as an infection with, e.g., VRE, C. difficile,Escherichia coli, Salmonella, Shigella, Campylobacter, Vibrio cholera,Clostridium perfringes, Bacillus cereus, Vibrio parahemolyticus,Yersinia enterocolitica, Helicobacter pylori, rotavirus, or norovirus.

In some embodiments, the GI tract infection is a fungal infection, suchas an infection with, e.g., Candida, Aspergillus, Mucor, Cryptococcus,Histoplasma, or Coccidioides.

In some embodiments, the GI tract infection is a protozoal infection,such as an infection with, e.g., Entamoeba histolytica, Giardia lamblia,Cryptosporidium parvum.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingdiarrhea, such as, e.g., Clostridium difficile associated diarrhea(CDAD), antibiotic-associated diarrhea (AAD), antibiotic-induceddiarrhea, travellers' diarrhea (TD), pediatric diarrhea, or (acute)infectious diarrhea.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingnecrotizing enterocolitis (NEC); gastroenteritis; small intestinebacterial overgrowth (SIBO) or similar disease, disorder or conditionassociated with a GI tract infection.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of having coloncancer, liver cancer, ameboma; indigestion or non-ulcer dyspepsia; analfissures, perianal abscess and anal fistula; diverticulosis ordiverticulitis; peptic ulcer or similar disease, disorder or conditionassociated with structural alterations of the GI tract.

In some embodiments, subjects with Clostridium difficile infection(CDI)-induced colitis may be treated according to the methods providedherein. Subjects with CDI-induced colitis may present with waterydiarrhea, cramping, abdominal pain, anorexia, malaise, fever,dehydration, lower abdominal tenderness, and/or rebound tenderness. Thepresence of C. difficile in the stool of patients can be tested by stoolculture, glutamate dehydrogenase enzyme immunoassay, PCR assay to detectgenes for C. difficile toxins, stool cytotoxin assay, or enzymeimmunoassay for C. difficile toxins A and B. Patient populations includesubjects with primary CDI, subjects with recurrent CDI, subjects withdifferent severities of CDI-associated diarrhea (mild, moderate,severe), and subjects at risk for CDI due to the presence of riskfactors such as antibiotics treatment, broad-spectrum antibioticstreatment, residence in a hospital or long-term care facility,gastrointestinal tract surgery, diseases of the colon, a weakened immunesystem, chemotherapy, advanced age, kidney disease, or use ofproton-pump inhibitors. Standard-of-care treatments for CDI includeantibiotics such as metronidazole, fidaxomicin, or vancomycin.Treatments may also include probiotics, fecal transplant, and fluids toprevent dehydration. Resolution of disease is measured by abatement ofdiarrhea (e.g., the absence of a 24 hour period with more than threeunformed stools) and resolution of other symptoms described above.Clearance of infection may be verified by the absence of a positivestool test for C. difficile.

In one embodiment, methods are provided to prevent, treat, amelioratesymptoms of, and/or prevent initial colonization or relapse ofcolonization by pathogens. In some embodiments, the relapse occursduring or after first-line or standard-of-care treatment regimen. Insome cases, a pathogen load may initially lighten upon thestandard-of-care treatment but then the load begins to increase again,potentially triggering a relapse of the disease. In some embodiments,glycan therapeutics may be administered (e.g. at the beginning, duringor after the initial treatment regimen) to prevent the relapse or treatone or more relapse symptoms. In some embodiments, disease-associatedbacteria, pathobionts or pathogens are selected from the groupconsisting of the species Bilophila wadsworthia, Campylobacter jejuni,Citrobacter farmer, Clostridium difficile, Clostridium perfringens,Clostridium tetani, Collinsella aerofaciens, Enterobacter hormaechei,Enterococcus faecalis, Enterococcus faecium, Escherichia coli,Fusobacterium varium, Fusobacterium nucleatum, Haemophilusparainfluenzae, Klebsiella pneumonia, Peptostreptococcus stomatis,Porphyromonas asaccharolytica, Pseudomonas aeruginosa, Salmonellabongori, Salmonella enteric, Shigella boydii, Shigella dysenteriae,Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Streptococcusinfantarius, Vibrio cholera, and Yersinia enterocolitica.

In some embodiments, disease-associated bacteria, pathobionts orpathogens include the genera Bilophila, Campylobacter, Candidatus,Citrobacter, Clostridium, Collinsella, Desulfovibrio, Enterobacter,Enterococcus, Escherichia, Fusobacterium, Haemophilus, Klebsiella,Lachnospiraceae, Peptostreptococcus, Porphyromonas, Portiera,Providencia, Pseudomonas, Salmonella, Shigella, Staphylococcus,Streptococcus, Vibrio, and Yersinia.

In one embodiment, provided herein is a method of preventing relapse ofC. difficile symptoms in a subject having been treated with a first-linedrug (e.g. vancomycin, metronidazole, fidaxomicin). The method includesthe steps of identifying a subject infected with C. difficile and havingbeen administered an antibiotic and administering to the subject apharmaceutical composition comprising a glycan therapeutic in an amounteffective to prevent the recurrence of one or more symptoms associatedwith C. difficile infection. In some embodiments, viable C. difficilepathogen is retained in the gastrointestinal tract of the subject (e.g.CFU counts are detectable in a sample taken from the subject, e.g. afecal sample) even post-treatment with the antibiotic but C. difficileassociated symptoms are significantly reduced.

In some embodiments, subjects exhibiting vancomycin-resistantenterococci (VRE) colonization and infection may be treated according tothe methods provided herein. Bacteria of the genus Enterococcus arecommon members of the gut microbiota. Vancomycin-resistant members ofthis genus, commonly E. faecalis and E. faecium, can causevancomycin-resistant enterococci (VRE) colonization and infection.Subjects colonized with VRE may present with a VRE-positive stoolsample, rectal swab, perirectal swab, or sample from another body site.Vancomycin resistance can be assessed by bacterial culture or byPCR-based assays that detect vancomycin resistance (Van) gene operons.Although colonized subjects may be asymptomatic, this population is atincreased risk for infection with VRE. Subjects with VRE infection maypresent with diarrhea, fever, chills, urinary tract infection (UTI),bacteremia, endocarditis, intra-abdominal and pelvic infection,respiratory infection, or infection at another body site. Patientpopulations include subjects who are colonized with VRE, subjectssuffering from a VRE infection, and subjects who are at risk forcolonization or infection with VRE due to the presence of risk factorssuch as hospitalization, residence in a long-term care facility,long-term antibiotic use, immunosuppression, surgery, open wounds,indwelling devices (e.g., intravenous lines or urinary catheters), oremployment as a health care worker. Standard prevention measures for VREcolonization or infection include strict adherence to good hygienepractices (e.g., hand washing) and avoidance of risk factors wherepossible (e.g., removal of indwelling devices). Subjects colonized withVRE but not suffering from a VRE infection are typically not treated.Standard-of-care treatment options for VRE infections are limited due toresistance to standard antibiotics, but can include combinations ofantibiotics and/or antibiotics such as quinupristin-dalfopristin,linezolid, daptomycin, and tigecycline that have been demonstrated toretain activity against many strains of VRE. Treatments may also includeprobiotics or supportive care. Resolution of disease is measured byclearance of infection and resolution of other symptoms described above.Clearance of infection or colonization may be verified by the absence ofa VRE-positive test in a relevant biological sample. Prevention ofinfection or colonization may be quantified in a similar manner.

Inflammatory Diseases

In some embodiments, administration of the glycan therapeutic reducesinflammation. In some embodiments, a subject is identified to besuitable for treatment if the subject has or is suspected of having adisease, disorder or condition including: gastrointestinal inflammatorydiseases including inflammatory bowel disease (IBD), ulcerative colitis(UC), Crohn's disease (CD), idiopathic inflammation of the small bowel,indeterminatal colitis, pouchitis; irritable bowel syndrome (IBS), colonand liver cancers, necrotizing enterocolitis (NEC), intestinalinflammation, constipation, microscopic colitis, diarrhea; graft versushost disease (GVHD); (food) allergies; pseudomembranous colitis;indigestion or non-ulcer dyspepsia; diverticulosis or diverticulitis,ischemic colitis; radiation colitis or enteritis; collagenous colitis;gastroenteritis; and polyps.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havinginflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn'sdisease (CD), intestinal inflammation, microscopic colitis or similardisease, disorder or condition that is associated with inflammation ofthe intestine.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingidiopathic inflammation of the small bowel, indeterminatal colitis,pouchitis, pseudomembranous colitis, ischemic colitis, radiation colitis(enteritis), collagenous colitis or similar disease, disorder orcondition that is associated with inflammation of the intestine.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havinggastroenteritis; graft versus host disease (GVHD), or a (food) allergy.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingirritable bowel syndrome (IBS), constipation, diarrhea, indigestion,non-ulcer dyspepsia or similar disease, disorder or condition that isassociated with an altered intestinal transit.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of having coloncancer, liver cancers, necrotizing enterocolitis (NEC); diverticulosisor diverticulitis; polyps or similar disease, disorder or condition thatis associated with structural alteration of the intestine.

Subjects with inflammatory bowel disease (IBD) may present withabdominal cramps and pain, diarrhea that may be bloody, urgency of bowelmovements, constipation, nausea, vomiting, fever, weight loss, loss ofappetite, and/or iron deficiency anemia due to blood loss. Symptoms ofIBD may occur in flares, with alternating periods of symptomatic andasymptomatic disease. IBD may be diagnosed by a combination of tests,including stool exams (to eliminate the possibility of infectious causesof diarrhea, check for trace amounts of blood in the stool, and quantifybiomarkers associated with IBD such as fecal calprotectin), a completeblood count to assess levels of inflammation, blood tests to assessbiomarkers including C-reactive protein (CRP) and perinuclearanti-neutrophil cytoplasmic antibody (pANCA), barium X-ray,sigmoidoscopy, colonoscopy, and endoscopy. Patient populations includesubjects with ulcerative colitis (UC; limited to the colon or largeintestine), subjects with Crohn's disease (CD; affecting any segment ofthe gastrointestinal tract), and subjects with different diseaseseverities (mild, moderate, severe). Standard-of-care treatments for IBDinclude aminosalicylates (e.g., sulfasalazine, mesalamine, balsalazide,olsalazine), corticosteroids (e.g., hydrocortisone, prednisone,methylprednisolone, prednisolone, budesonide, dexamethasone),immunosuppressants (e.g., azathioprine, 6-mercaptopurine, methotrexate,cyclosporine), antibiotics (e.g., metronidazole, ciprofloxacin,rifaximin), tumor necrosis factor inhibitors (e.g, infliximab,adalimumab, certolizumab pegol), integrin inhibitors (e.g., natalizumab,vedolizumab), and surgery. Resolution or control of disease may bequantified by endoscopic or sigmoidoscopic assessment of diseaseseverity according to standard scoring metrics, abatement of symptomsdescribed above, reduction in disease severity as determined bycomposite indexes such as the Crohn's Disease Activity Index (CDAI), orimprovement in health-related quality of life as measured by the IBDQuestionnaire (IBD-Q).

Metabolic Diseases

In some embodiments, a subject is identified to be suitable fortreatment if the subject has or is suspected of having a disease,disorder or condition including: obesity, pre-diabetes, type IIdiabetes, high blood cholesterol, high LDL, high blood pressure, highfasting blood sugar, high triglyceride levels, low HDL non-alcoholicfatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH);metabolic syndrome; hyperammonemia, essential nutrient deficiency,hemochromatosis, lactose intolerance, gluten intolerance; andacrodermatitis enteropathica.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingobesity, (insulin resistance) pre-diabetes, type II diabetes, highfasting blood sugar (hyperglycemia), metabolic syndrome or similardisease, disorder or condition associated with metabolic diseasesymptoms.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of having highblood cholesterol, high LDL, high blood pressure (hypertension), hightriglyceride levels, low HDL or similar cardiovascular risk factor.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingnon-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis(NASH), hyperammonemia or similar disease, disorder or condition of theliver.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havinglactose intolerance, gluten intolerance or similar disease, disorder orcondition that is associated with food intolerance.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingessential nutrient deficiency, hemochromatosis, acrodermatitisenteropathica or similar disease, disorder or condition that isassociated with a nutrient mismanagement.

In one embodiment, provided is a method of treating a metabolic disorderin a human in need thereof, by: administering to the human apharmaceutical glycan therapeutic composition to treat the metabolicdisorder. In one embodiment, the metabolic disorder is selected fromobesity, adiposity, insulin resistance, diabetes, and fatty liversyndrome.

Metabolic disorders may include disorders, diseases, and conditions thatare caused or characterized by abnormal weight gain; energy use orconsumption; altered responses to nutrients, energy sources, hormones,or other signaling molecules; or altered metabolism of carbohydrates,lipids, proteins, or nucleic acids, or a combination thereof. Examplesof metabolic disorders include insulin resistance, insulin sensitivity,fatty liver syndrome, obesity, adiposity, and diabetes (e.g., type 1diabetes, type 2 diabetes). In one variation, the methods providedherein treat obesity. Provided herein are methods for treating obesityin a subject in need thereof using a pharmaceutical glycan therapeuticcomposition that can alter gut microbiota of the subject in a way thatresults in weight loss and/or decreased body fat in the subject.

In one embodiment, provided is a method of reducing adiposity in asubject in need thereof, by: administering to the human a pharmaceuticalglycan therapeutic composition in an amount effective to reduceadiposity. Adiposity may be determined using any appropriate methodknown in the art, including, for example, waist circumference, waist tohip ratio, skinfold thickness, bioelectric impedance, underwaterweighing, air-displacement plethysmography, or hydrometry.

In one embodiment, provided is a method of improving glucose metabolismin a subject in need thereof, by: administering to the subject apharmaceutical glycan therapeutic composition in an amount effective toimprove glucose metabolism. Glucose metabolism may be determined by anyappropriate method known in the art, including, for example, fastingblood sugar level, fasting insulin level, postprandial blood sugar test,postprandial insulin test, oral glucose tolerance test, intravenousglucose tolerance test, glycated hemoglobin level, or random blood sugartest.

In one embodiment, provided is a method of increasing insulinsensitivity in a human, by: administering to the subject apharmaceutical glycan therapeutic composition in an amount effective toincrease insulin sensitivity, wherein the human has an insulinsensitivity prior to the administration of the glycan therapeutic and aninsulin sensitivity after the administration of the glycan therapeutic,and the insulin sensitivity of the human after the administration of theglycan therapeutic is higher than the insulin sensitivity of the humanprior to the administration of the glycan therapeutic. Insulinsensitivity may be determined by any appropriate method known in theart, including, for example, fasting blood sugar level, fasting insulinlevel, postprandial blood sugar test, postprandial insulin test, oralglucose tolerance test, intravenous glucose tolerance test, glycatedhemoglobin level, or random blood sugar test.

In some embodiments, subjects with type 2 diabetes may be treatedaccording to the methods provided herein. Subjects with type 2 diabetesmay present with blurred vision, peripheral neuropathy, increasedurination, increased thirst, fatigue, increased hunger, weight loss, oryeast, bladder, kidney, skin, or other infections. Type 2 diabetes isdiagnosed by criteria described by the American Diabetes Association(ADA), including the following: fasting plasma glucose (FPG) of 126mg/dL (7 mM) or higher, or a 2 hour plasma glucose level of 200 mg/dL(11.1 mM) or higher during a 75 g oral glucose tolerance test (OGTT), ora random plasma glucose of 200 mg/dL (11.1 mM) or higher in a patientwith classic symptoms of hyperglycemia or hyperglycemic crisis, or ahemoglobin A1c (HbA1c) level of 6.5% or higher. Patient populationsinclude adults and children with type 2 diabetes, subjects at risk fordeveloping type 2 diabetes (e.g., subjects with prediabetes or subjectswho are overweight), and subjects with type 2 diabetes in conjunctionwith conditions of metabolic syndrome including obesity, elevated bloodpressure, elevated serum triglycerides, and low high-density lipoprotein(HDL) levels. Standard-of-care treatments for type 2 diabetes includelifestyle management (diet, exercise, and behavioral modifications),alpha-glucosidase inhibitors, biguanides (e.g., metformin),sulfonylureas, dipeptidyl peptidase IV (DPP-4) inhibitors, glucagon-likepeptide-1 (GLP-1) analogs, meglitinides, selective sodium-glucosetransporter-2 (SGLT2) inhibitors, thiazolidinediones, insulin, andamylinomimetics. Treatment efficacy may be assessed by resolution of thesymptoms or diagnostic criteria listed above (e.g., decrease in FPG tohealthy levels), or, in subjects at risk for developing type 2 diabetes,by decreased rates of conversion to a type 2 diabetic state.

In some embodiments, subjects exhibiting Non-alcoholic fatty liverdisease (NAFLD) and/or non-alcoholic steatohepatitis (NASH) may betreated according to the methods provided herein. Non-alcoholic fattyliver disease (NAFLD) is characterized by an abnormal buildup of fat inthe liver. NAFLD can progress to non-alcoholic steatohepatitis (NASH),which is characterized by liver inflammation, fibrosis, and cirrhosis.Subjects with NAFLD may be asymptomatic. Subjects with NAFLD or NASH maypresent with increased liver size (noted during physical exam), fatigue,weight loss, general weakness, and/or ache in the upper right of thebelly. Diagnosis of NAFLD/NASH includes elevated blood levels of alanineaminotransferase (ALT) or aspartate aminotransferase (AST), enlargedliver and specific histopathologic markers (e.g. by liver biopsy,abdominal ultrasound, CT scan, or an MRI scan). Patient populationsinclude subjects with NAFLD, subjects with NASH, subjects at risk ofdeveloping NAFLD/NASH (e.g., subjects who are overweight or haveelevated cholesterol levels), and subjects with NAFLD/NASH inconjunction with conditions of metabolic syndrome including obesity,elevated fasting plasma glucose, elevated blood pressure, elevated serumtriglycerides, and low high-density lipoprotein (HDL) levels.Standard-of-care treatments for NAFLD/NASH include lifestyle management(diet, exercise, behavioral modifications, and avoidance of alcohol).

Treatments in clinical trials or under development include farnesoid Xreceptor (FXR) agonists (e.g., obeticholic acid), Takeda Gprotein-coupled receptor 5 (TGR5) agonists, fatty acid-bile acidconjugates (e.g., aramchol), antioxidants (e.g., vitamin E),antifibrotic agents, peroxisome proliferator-activated receptor(PPAR)-gamma agonists, PPAR alpha/delta agonists, caspase inhibitors(e.g., Emricasan), and/or galectin-3 inhibitors. Treatment efficacy maybe assessed by resolution of the symptoms or diagnostic criteria listedabove (e.g., decrease in ALT to healthy levels), or, in subjects at riskfor developing NAFLD/NASH, by decreased rates of conversion toNAFLD/NASH.

In some embodiments, obese subjects may be treated according to themethods provided herein. Obesity is a significant health concern, andmay have a negative effect on health. For example, obesity may lead toreduced life expectancy and/or increased health problems, such asdiabetes, high blood pressure, heart disease, stroke, high cholesterol,sleep apnea, and arthritis. Obese subjects present with a body massindex (BMI) of greater than 30 kg/m². Alternatively, obese subjects maybe classified based on body fat percentage (greater than 25% for malesor greater than 33% for females). Diagnosis may also include anevaluation of fasting lipid levels (cholesterol, triglycerides), liverfunction, glucose levels, insulin levels, glycosylated hemoglobin(HbA1c), and/or glucose tolerance. Patient populations include subjectswith childhood obesity, moderate obesity, morbid/severe obesity, geneticcauses of obesity (including Prader-Willi syndrome, Bardet-Biedlsyndrome, Cohen syndrome, and MOMO syndrome), and obesity in conjunctionwith other conditions of metabolic syndrome (elevated blood pressure,elevated fasting plasma glucose, elevated serum triglycerides, and lowhigh-density lipoprotein (HDL) levels). Standard-of-care treatments forobesity include lifestyle management (diet, exercise, and behavioralmodifications), bariatric surgery, medications that impair dietaryabsorption (e.g., tetrahydrolipstatin), medications that impair dietaryintake, medications that increase energy expenditure, and medications totreat common comorbidities (e.g., medications for type 2 diabetes orhypertension). Treatment endpoints include change in body weight,fasting lipid levels, liver function, glucose levels, insulin levels,HbA1C, and/or glucose tolerance.

Other Diseases

In some embodiments, a subject is identified to be suitable fortreatment if the subject has or is suspected of having a disease,disorder or condition including: autoimmune arthritis, type I diabetes,atopic dermatitis, autism, asthma, cardiovascular disease, chronickidney disease, multiple sclerosis, heart disease, psoriasis,hyperammonemia, hepatic encephalopathy, cachexia, Gout, drug intolerance(e.g., to metformin), low oral bioavailability of drugs, fecalincontinence, Hirschsprung's disease, anismus, colic, ileus,hemorrhoids, and intussusceptions.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingautoimmune arthritis, type I diabetes, multiple sclerosis, psoriasis orsimilar autoimmune disease, disorder or condition.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingasthma, atopic dermatitis or similar environmental-driven allergy.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingchronic kidney disease, heart disease, cardiovascular disease or similardisease, disorder or condition that is associated with organ failure.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingautism, hyperammonemia, hepatic encephalopathy or similar disease,disorder or condition that is associated with neurological symptoms.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingcachexia, Gout or similar nutritional disorder.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingHirschsprung's disease, ileus, anismus, intussusceptions, fecalincontinence, hemorrhoids or similar gastrointestinal disorder.

In some embodiments, subjects with atopic dermatitis (AD) may be treatedaccording to the methods provided herein. Subjects with atopicdermatitis (AD) may present with skin that is dry, itchy, and/orinflamed. Diagnosis and severity of AD may be determined by using theSCORAD index (Oranje, A. P., et al. “Practical issues on interpretationof scoring atopic dermatitis: the SCORAD index, objective SCORAD and thethree-item severity score.” British Journal of Dermatology 157.4 (2007):645-648) or the Eczema Area and Severity Index (EASI) score (Hanifin etal., The eczema area and severity index (EASI): assessment ofreliability in atopic dermatitis, Experimental Dermatology, 2001,10:11). AD may occur in flares, with alternating periods of symptomaticand asymptomatic disease. Staphylococcus aureus is commonly present onskin sites with AD, and biomarkers including IgE and inflammatory or Th2cytokines and chemokines may also be elevated in the diseased skin orsystemically. Patient populations include infants with early-onset AD,children with pediatric AD, adults with late-onset AD, pregnant women atrisk for flares of AD (“atopic eruption of pregnancy”), subjects withmild, moderate, or severe AD flares, or subjects who are at risk ofdeveloping AD. Standard-of-care treatments for AD include topicallyapplied moisturizers, topically applied steroid ointments such ashydrocortisone, bleach baths, antibiotics, immunomodulatory agents suchas tacrolimus, antihistamines, antibody-based therapies (includingantibodies to block IgE, the IL-4 receptor, IL-4, and IL-13), and otheranti-inflammatory agents. Treatment may also include probiotics.Resolution or control of disease may be quantified by the standardSCORAD or EASI criteria described above.

In some embodiments, subjects with asthma may be treated according tothe methods provided herein. Subjects with asthma may present withwheezing, coughing, shortness of breath, and/or chest tightness or pain.These symptoms are commonly episodic and may be triggered by factorssuch as exercise or exposure to allergens. Additionally, children withasthma may present with a history of recurrent bronchitis,bronchiolitis, or pneumonia or a persistent cough with colds. Diagnosisof asthma is established by lung function testing with spirometry in thepresence and absence of treatment with a bronchodilator. Patientpopulations include infants with asthma; subjects with childhood asthma;adult-onset asthma; intermittent, mild persistent, moderate persistent,or severe persistent asthma; exercise-induced asthma; allergic asthma;cough-variant asthma; occupational asthma; nocturnal asthma; andsubjects who are at risk of developing asthma, for example, due to afamily history of atopy. Standard-of-care treatments for asthma includeinhaled corticosteroids (e.g., budesonide, fluticasone, beclomethasone,mometasone, and ciclesonide), short-acting bronchodilators (e.g.,albuterol), long-acting bronchodilators (e.g., salmeterol), leukotrienemodifiers (e.g., montelukast) or other anti-inflammatory agents,anti-cholinergic agents (e.g., ipratropium, tiotropium), anti-IgE (e.g.,omalizumab) for allergic asthma, and/or systemic steroids (e.g.,prednisone, prednisolone, methylprednisolone, dexamethasone). Treatmentsmay also include probiotics. Treatment efficacy may be assessed by adecrease in the frequency or severity of the symptoms described above,improvement in lung function (assessed by measurements such as peakexpiratory flow rate (PEFR) or forced expiratory volume in 1 second(FEV1)), decrease in the need to continue or initiate treatments forasthma, or changes in the levels of biomarkers of airway inflammation(e.g., serum IgE, exhaled nitric oxide, sputum or blood eosinophilcounts, inflammatory cytokines, Th2 cytokines, etc.).

In some embodiments, subjects with chronic kidney disease (CKD) may betreated according to the methods provided herein. Subjects with CKD maypresent with fatigue, trouble concentrating, poor appetite, troublesleeping, nocturnal muscle cramping, swollen feet and ankles, skinrash/itching, nausea, vomiting, a metallic taste in the mouth, shortnessof breath, and/or increased urination. Diagnosis of kidney disease,including CKD, is performed by tests of the glomerular filtration rate(GFR), blood levels of urea and creatinine, urine levels of albumin,kidney biopsy, ultrasound, and/or CT scan. Patient populations includesubjects with CKD caused by diabetic nephropathy; subjects with CKDcaused by high blood pressure; subjects with polycystic kidney disease,pyelonephritis, or glomerulonephritis; subjects with kidney damage dueto long-term use of kidney-damaging medicines; and subjects at risk ofdeveloping CKD due to the presence of risk factors such as diabetes,high blood pressure, or family history of kidney disease.Standard-of-care treatments for CKD include medicines to lower bloodpressure, control blood glucose, and lower blood cholesterol. Treatmentsmay also include dietary modifications and probiotics. Treatmentefficacy may be assessed by resolution of the symptoms or diagnosticcriteria listed above (e.g., decrease in urine albumin and serumcreatinine), reduction in the need to start dialysis or prolongation ofthe time before starting dialysis, reduction in blood levels of uremicsolutes (e.g., p-cresol sulfate and indoxyl sulfate) or otherpotentially harmful circulating factors (e.g., trimethylamine N-oxide(TMAO), or, in subjects at risk for developing CKD, by decreased ratesof conversion to CKD.

In some embodiments, subjects with Hepatic encephalopathy (HE) may betreated according to the methods provided herein. Hepatic encephalopathyincludes multiple adverse neurological symptoms that occur when theliver is unable to remove toxic substances such as ammonia from theblood. Subjects with HE may present with confusion, forgetfulness,anxiety or excitation, sudden changes in personality or behavior,changes in sleep patterns, disorientation, sweet or musty smellingbreath, slurred speech, and/or difficulty controlling motor functions.Diagnosis of HE is performed by tests of liver function, serum ammonialevels, EEG, and other blood and neurological tests. Patient populationsinclude subjects with mild HE, severe HE, overt HE, subjects who havepreviously experience one or more episodes of HE, and patients who areat risk for HE due to the presence of risk factors such as liver damage.Standard-of-care treatments for HE include lactulose, lactitol, andantibiotics (e.g., rifaximin or neomycin). Treatments may also includedietary modifications and probiotics. Treatment efficacy may be assessedby resolution of the symptoms or diagnostic criteria listed above (e.g.,reduction in serum ammonia levels), decreased incidence of futureepisodes of HE, or, in subjects at risk of HE, by decreased occurrenceof an initial episode of HE.

Drug- or Treatment-Induced Digestive Abnormalities

Provided herein are methods of reducing drug- or treatment-inducedsymptoms in a human subject. Such drug- or treatment-induced symptomsinclude any digestive abnormalities. Exemplary digestive abnormaliesinclude, but are not limited to weight-gain, constipation, heartburn,upset stomach, gas, bloating, flatulence, diarrhea, abdominal pain,cramping, nausea, and vomiting. In some embodiments, the digestiveabnormality is diarrhea. The method include administering to the humansubject a pharmaceutical composition comprising a glycan therapeuticpreparation in an amount effective to reduce one or more symptomsinduced by a drug or treatment. In one embodiment, the treatment isradiation treatment.

In one embodiment, the subject being identified to be suitable fortreatment with a glycan therapeutic has or is suspected of havingdrug-induced diarrhea, drug-induced constipation, drug-induced toxicity,drug-induced intolerance (e.g. to metformin, to chemotherapies),drug-induced microbiome damage, drug-induced microbiome disease,drug-induced gastrointestinal disease, drug-induced enteritis or colitisor similar drug-induced disorder or condition.

In some embodiments, the pharmaceutical composition comprising a glycantherapeutic preparation is administered prior to, concomitant with orafter administration of the drug (or radiation treatment),administration of which induces the symptoms. Exemplary drugs whichoften are associated with drug- or treatment-induced symptoms include,but are not limited to a cancer drug, an anti-diabetic, animmune-suppressive drug, an antimicrobial drug, a chemotherapeutic, ananti-psychotic, a proton pump inhibitor, and a non-steroidanti-inflammatory drug (NSAID). Administration of these drugs generallyis associated with dysbioses that can, e.g., occur during the treatmentregimen. In some embodiments, the dysbiosis causes or amplifies thedrug- or treatment-induced symptoms, such as digestive abnormalities. Insome embodiments, administration of the glycan therapeutic modulates themicrobiome such that the drug- or treatment-induced symptoms arereduced. In some embodiments, the glycan therapeutic promotes the growthof commensal bacteria and/or supports the growth of beneficial microbialcommunities which would negatively be affected or lost in response tothe drug treatment or which can complement commensal bacteria that havebeen negatively affected or lost in response to the drug treatment.

Specific examples of drugs associated with digestive abnormalitiessymptoms of which can be reduced by administration of the glycantherapeutic include, but are not limited to ciprofloxacin, clindamycin,amoxicillin-clavulanate, cefixime, ephalosporins, fluoroquinolones,azithromycin, clarithromycin, erythromycin, tetracycline, azithromycin,irinotecan (camptosar), 5-fluorouracil, leucovorin, oxaliplatin,bortezomib, imatinib, lenalidomide, imbruvica, ipilimumab, pertuzumab,capecitabine, docetaxel, lapatinib, erlotinib, carmustine, etoposide,aracytine, melphalan, cytarabine, daunorubicine, amsacrine,mitoxantrone, olanzapine, ranitidine, famotidine, cimetidine,omeprazole, sucralfate, esomeprazole, naproxen, diclofenac,indomethacin, ibuprofen, ketoprofen, piroxicam, celecoxib, nimesulid,aspirin, metformin, paroxetine, valproic acid, or clozapine.

In some embodiments, the digestive abnormalities are associated withtreatment of the subject with a chemotherapeutic agent. In oneembodiment, the digestive abnormality is diarrhea. In specificembodiments, the chemotherapeutic agent is Irinotecan, 5-fluorouracil,leucovorin, or combinations thereof. In specific embodiments, thechemotherapeutic agent is oxaliplatin, leucovorin, 5-fluorouracil, orcombinations thereof. In specific embodiments the chemotherapeutic agentis bortezomib, imatinib, lenalidomide, imbruvica, ipilimumab,pertuzumab, capecitabine, docetaxel, lapatinib, erlotinib, orcombinations thereof. In some embodiments, the chemotherapeutic agent isCarmustine, Etoposide, Aracytine, Melphalan, or combinations thereof. Inspecific embodiments the chemotherapeutic agent is cytarabine,daunorubicine, etoposide, or combinations thereof. In specificembodiments the chemotherapeutic agent is amsacrine, cytarabine,etoposide, or combinations thereof. In specific embodiments, thechemotherapeutic agent is mitoxantrone, cytarabine, or combinationsthereof.

In some embodiments, the digestive abnormalities are associated withtreatment of the subject with an antibiotic. In one embodiment, thedigestive abnormality is diarrhea. In specific embodiments, theantibiotic is ciprofloxacin, clindamycin, amoxicillin-clavulanate,cefixime, ephalosporins, fluoroquinolones, azithromycin, clarithromycin,erythromycin, tetracycline, or azithromycin.

In some embodiments, the digestive abnormalities are associated withtreatment of the subject with an anti-psychotic drug. In one embodiment,the digestive abnormality is weight gain. In one embodiment, the drug isolanzapine.

In some embodiments, the digestive abnormalities are associated withtreatment of the subject with a proton-pump inhibitor drug. In oneembodiment, the digestive abnormality is diarrhea. In specificembodiments, the drug is ranitidine, famotidine, cimetidine, omeprazole,sucralfate, or esomeprazole.

In some embodiments, the digestive abnormalities are associated withtreatment of the subject with a non-steroidal anti-inflammatory drug(NSAID). In one embodiment, the digestive abnormality is diarrhea. Inspecific embodiments, the drug is naproxen, diclofenac, indomethacin,ibuprofen, ketoprofen, piroxicam, celecoxib, nimesulid, or aspirin.

In some embodiments, the digestive abnormalities are associated withtreatment of the subject with metformin, paroxetine, valproic acid, orclozapine.

In one embodiment, reducing the one or more symptoms increasescompliance by the subject to the treatment regimen. In one embodiment,reducing one or more symptom enables the physician to prescribe ahigher-dose of the drug to be administered. In such embodiments,treatment of the underlying disease is more effective (e.g. increasedreduction of symptoms, shorter period to achieve a disease orsymptom-free state, or longer maintenance of a disease or symptom-freestate, etc.).

Other Embodiments

In some embodiments, the subject experiences a reduction in at least onesymptom of the gastrointestinal disease, disorder or condition followingtreatment. In some embodiments, a reduction in the severity of a symptomfollowing treatment can be determined (e.g. by measuring a knownbiomarker) and is in the order of about 3%, 5%, 7%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, or about 100%. In some embodiments, thesymptoms, measured as described herein, are decreased by an average ofabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100%when compared to symptoms prior to the administration of apharmaceutical glycan therapeutic composition. In some embodiments, thereduction in the severity of the symptom persists for at least about aday, two days, three days, four days, five days, a week, two weeks,three weeks, a month, 3 months, 6 months, 9 months, a year, two years,five years, ten years after treatment or the reduction is permanent.

In one embodiment, a symptom of a gastrointestinal disease, disorder orcondition remains partially, substantially, or completely eliminated ordecreased in severity in a subject for at least about 1 day, 1 week, 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, oneyear, 18 months, two years, three years, four years, five years, tenyears, or more than ten years after the termination of treatment. Inanother embodiment a symptom of a gastrointestinal disease, disorder orcondition is permanently eliminated or decreased in severity in asubject after the termination of treatment.

In some embodiments, administration of the pharmaceutical glycantherapeutic compositions improves the overall health of the host and/orthe health of a specific niche, such as the GI tract, e.g. by modulating(e.g. increasing or decreasing) the growth or abundance of one or moremembers of the microbial community in the niche (such as residentcommensal bacteria and/or acquired pathogens or pathobionts).

Research from the gut has led to the identification of biomarkers withthe potential to demonstrate the health effects of prebiotics, which mayalso be used to characterize the health effects and treatment efficaciesof the pharmaceutical glycan therapeutic compositions described hereinon the gastrointestinal microbiota and environment. These markersinclude: i) changes in gastrointestinal microbiota and the overallmetabolism of the gastric environment, such as the production of organicacids, ii) modulation of the immune system, assessing inflammatory andimmune globulins iii) increase the absorption of minerals in the colon,such as calcium, zinc or magnesium iv) regulation of lipid metabolism,lowering cholesterol, v) induction of other important processes for hosthomeostasis (see, reviews by Pool-Zobel B L. Inulin-type fructans andreduction in colon cancer risk: review of experimental and human data.2005. British Journal of Nutrition 93 Suppl 1:S73-90; and Liong M T.Roles of Probiotics and Prebiotics in Colon Cancer Prevention:Postulated Mechanisms and In-vivo Evidence.2008. International Journalof Molecular Sciences 9(5):854-63).

The pharmaceutical glycan therapeutic compositions when administered toa subject in an effective amount may modulate one or more host pathways.The glycan therapeutic treatment may result in increases or decreases ofone or more biomarkers that can be determined by methods known in theart. An investigator can easily determine at which point or pointsduring treatment the biomarker(s) should be measured, e.g. prior totreatment, at various intervals during treatment and/or after treatment.Any suitable sample, e.g. a gastrointestinal-specific sample such as,e.g. a tissue sample or biopsy, a swab, a gastrointestinal secretion(such as feces/a stool sample), etc. may be drawn from the subject andthe sample may be analyzed. In some embodiments, a substantial increaseor decrease in a biomarker may be detected.

In some embodiments, the glycan therapeutic is digested by the gutmicrobiota (e.g. Clostridia), resulting, e.g., in the release ofshort-chain fatty acids such as butyrate, acetate, and propionate, whichmay act in an immunomodulatory capacity (e.g. anti-inflammatory) andother metabolites (e.g. bile acids, and lactate) that may conferbeneficial health effects on the host.

To evaluate the effect of administered pharmaceutical glycan therapeuticcompositions on SCFA production in the gut, fecal samples can becollected. SCFA levels, particularly acetate, propionate, and butyratemay be quantified. SCFAs, creatines, and hydroxy-SCFAs can be quantifiedby alkalinizing stool samples, obtaining fingerprints of the metaboliccomposition of the sample using, e.g., 1D 1H NMR spectrometer, andanalyzing with supervised multivariate statistical methods. Inulin mayserve as a positive control.

In some embodiments, microbial metabolite profiles of patient samples ormicrobes cultures from subject samples are used to identify risk factorsfor developing a gastrointestinal infectious and/or inflammatorydisease, disorder or condition. Exemplary metabolites for the purposesof diagnosis, prognostic risk assessment, or treatment assessmentpurposes include those listed in Table 2. In some embodiments, microbialmetabolite profiles are taken at different time points during asubject's disease and treatment in order to better evaluate thesubject's disease state including recovery or relapse events. Suchmonitoring is also important to lower the risk of a subject developing anew gastrointestinal disease, disorder or condition. In someembodiments, metabolite profiles inform subsequent treatment.

Further, if determined useful by a treating physician or otherhealthcare provider, the pharmaceutical glycan therapeutic compositionsdescribed herein can be administered in combination with various otherstandard of care therapies. In some embodiments, the combination ofadministration of the glycan therapeutic and the standard-of-caretherapy agent has additive or synergistic treatment effects. Thepharmaceutical glycan therapeutic compositions may be administered priorto, concurrent with, or post treatment with standard of care therapies.In some instances, the therapies disrupt the composition and health ofthe GI tract's normal microbiota (e.g. use of anti-bacterial, anti-viralor anti-fungal agents), which may lead to the undesirable proliferationof harmful bacteria or pathogens, which may cause one or more of thesymptoms described herein. In some embodiments, administration of thepharmaceutical glycan therapeutic compositions described herein isuseful for alleviating those symptoms and improving the composition ofthe gastrointestinal microbial community.

Administration of Glycan Therapeutics

For any pharmaceutical glycan therapeutic composition used in a methoddescribed herein, a therapeutically effective dose can be estimatedinitially from laboratory animal models known to those of skill in theart. Such information can be used to more accurately determine usefuldoses in humans. Initial dosages can also be estimated from in vitro orin vivo data. Initial dosages can also be formulated by comparing theeffectiveness of the compounds used in the methods described herein inmodel assays with the effectiveness of known compounds. For instance,initial dosages can be formulated by comparing the effectiveness of theglycan therapeutic preparations in model assays with the effectivenessof other compounds that have shown efficacy in treating the presentconditions. In this method, an initial dosage can be obtained bymultiplying the ratio of effective concentrations obtained in the modelassay for the glycan therapeutic preparations used in methods describedherein and the control compound by the effective dosage of the controlcompound. For example, if a preparation useful in a present method istwice as effective in a model assay as a known compound (e.g., theefficacious concentration (EC₅₀) of the glycan therapeutic preparationis equal to one-half the EC₅₀ of the known compound in the same assay),an initial effective dosage of the glycan therapeutic preparation wouldbe one-half the known dosage for the known compound. Using these initialguidelines an effective dosage in subjects, such as humans, can bedetermined by one of ordinary skill. Dosage amount and interval may beadjusted individually to provide levels of the glycan therapeuticpreparation which are sufficient to maintain therapeutic effect. One ofskill in the art will be able to optimize therapeutically effectivelocal dosages without undue experimentation.

Depending upon the disorder and subject to be treated and the route ofadministration, the compositions may be administered at varying doses.In one embodiment, the smallest effective amount or dose of glycantherapeutic is used. In some embodiments, the glycan therapeutic isadministered in a dose from about 0.01 mg/kg to about 10,000 mg/kg, fromabout 0.1 mg/kg to about 1,000 mg/kg, from about 1 mg/kg to about 100mg/kg, 0.05 mg/kg to about 5,000 mg/kg, from about 0.5 mg/kg to about5,000 mg/kg, from about 5 mg/kg to about 500 mg/kg. This dose may begiven as mg/kg/day and may be administered as an initial dose or may beincreased or decreased over time (e.g., days or week) to reach a finaldose.

In some embodiments, the glycan therapeutic is administered in a totaldaily dose per subject from about 1 mg per day to about 100 grams perday; from about 10 mgs per day to about 10 grams per day; from about 100mgs per day to about 10 grams per day; from about 1 gram per day toabout 10 grams per day, from about 2 grams per day to about 20 grams perday; from about 5 grams per day to about 50 grams per day.

In some embodiments, a symptom of a gastrointestinal disease, disorderor condition in a subject exhibiting the symptoms is decreased oreliminated by administering to the subject increasing, decreasing orconstant amounts (or doses) of a pharmaceutical glycan therapeuticcomposition for a period of time (e.g. a treatment period).

In one embodiment, the composition contains beneficial, commensal and/orprobiotic bacterial strains in an amount comprised from 1×10⁷ to 1×10¹³CFU/dose and bacterial strain, or from 1×10⁹ to 1×10¹¹ CFU/dose andbacterial strain.

In some embodiments, the pharmaceutical composition is administered one,two, or three times a day. In some embodiments, the pharmaceuticalcomposition is administered twice a day. In some embodiments, thepharmaceutical composition is administered each day for a predeterminednumber of days (the treatment period). In some embodiments, thetreatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21,28, 35, 42, 49, 56, 63, 70, 100, 200, 300 or 365 days. In someembodiments, the treatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,or 12 months. In some embodiments, the treatment period is 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12 years, or life-long.

In one embodiment the total duration of treatment periods for agastrointestinal disease, disorder or condition can be from about oneday to 10 years, one day to 1 year, 1 day to 6 months, 1 day to 3months, 1 day to 1 months, one day to one week, one day to five days,one day to 10 days, one week to about 12 weeks, or about four weeks toabout ten weeks, or about four weeks to about eight weeks, or about sixweeks. The subject may undergo a suitable number of treatment periods,such as, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 treatmentperiods. During a treatment period, the subject takes a pharmaceuticalglycan therapeutic composition described herein, optionally along withingestion of prebiotic and/or probiotic containing food products. In oneembodiment, a pharmaceutical glycan therapeutic composition can also beadministered in combination with another substance (such as a probioticor commensal beneficial bacteria, a prebiotic substance or a therapeuticagent), as described herein.

In some embodiments, the pharmaceutical glycan therapeutic compositionmay also be combined with an antibiotic that disrupts normalgastrointestinal microbiota growth. Typically durations for antibiotictreatments are 1-14 days, or 2-10 days, or 5-7 days. In someembodiments, a glycan therapeutic is administered to a subject in needthereof immediately after one or more antibiotic treatment(s) has ended(e.g. 1 hour, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks or 4 weeks after theantibiotic treatment has ended). During a course of antibiotictreatment, the pharmaceutical glycan therapeutic composition may beprovided at the initiation of antibiotic treatment; shortly followingantibiotic treatment, e.g. 1, 2, 3, 4, 5, 6, 7, or more days followingtreatment; or may be administered upon diagnosis of undesirable pathogengrowth.

In some embodiments, the pharmaceutical glycan therapeutic compositionmay also be combined with a dysbiosis-causing drug, e.g. a drug thatdisrupts normal gastrointestinal microbiota growth, e.g. achemotherapeutic drug, an anti-diabetic drug, an immune-suppressivedrug, an antimicrobial drug, an anti-psychotic drug, a proton pumpinhibitor drug, or a non-steroid anti-inflammatory drug (NSAID). Thepharmaceutical glycan therapeutic composition, in some embodiments,reduces the drug- or treatment-induced symptoms in a human subject. Thesymptoms include digestive abnormalities, such as, e.g., weight-gain,constipation, heartburn, upset stomach, gas, bloating, flatulence,diarrhea, abdominal pain, cramping, nausea, and vomiting. In someembodiments, a glycan therapeutic is administered to a subject in needthereof immediately after one or more drug treatment(s) has ended (e.g.1 hour, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days,5 days, 6 days, 7 days, 2 weeks, 3 weeks or 4 weeks after the antibiotictreatment has ended). During a course of drug treatment, thepharmaceutical glycan therapeutic composition may be provided prior tothe initiation of drug treatment (e.g. 1, 2, 3, 4, 5, 6, 7 days prior);at the day of initiation of drug treatment; or shortly followingantibiotic treatment, e.g. 1, 2, 3, 4, 5, 6, 7, or more days followingtreatment, and may optionally be provided only initially (e.g. for ashort period) or throughout the duration of the drug-treatment, and mayeven be continued for a desired period after the drug treatment periodhas ended (e.g. for 1-7 days, 1-14 days, or 1-21 days thereafter). Insome embodiments, administration of the pharmaceutical glycantherapeutic composition is initiated or continued when one or moreadverse effects occur and/or are diagnosed (e.g. digestive abnormalitiesor pathogen growth) in conjunction with the drug treatment. In someembodiments, the treatment agent causing a dysbiosis is not a drug butradiation treatment or surgery and the pharmaceutical glycan therapeuticcomposition may also be administered as described herein.

In some embodiments, the total number and duration of treatment periodsis based on a subject's response to the treatment. For example, anindividual can experience a reduction in symptoms after 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 days of treatment with apharmaceutical glycan therapeutic composition. In another example, anindividual can experience a reduction in symptoms after 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12 months of treatment with a pharmaceutical glycantherapeutic composition. Thus, the duration of treatment is determinedby an individual subject's response to a pharmaceutical glycantherapeutic composition and the onset of relief from one or moresymptoms. Thus, a subject can experience symptoms at a given dose of apharmaceutical glycan therapeutic composition and can require that thesubject stay at that dose, or a lower dose, until symptoms subside.Thus, in one embodiment, the duration of the treatment is not determinedat the outset, but continues until the maximum dose of a pharmaceuticalglycan therapeutic composition is achieved per day, or until the desiredlevel of reduction in symptoms is achieved. In one embodiment, thetreatment is continuous.

In one embodiment, a subject can be given one dose for the firsttreatment period during a treatment regimen and a second dose during asecond treatment period. For example, a subject can be administered onedose of pharmaceutical glycan therapeutic composition for a one weekperiod and a second dose for a subsequent one week period.

A subject may self-administer a pharmaceutical glycan therapeuticcomposition and the glycan therapeutic composition is supplied orrecommended (or prescribed) by a health professional, e.g., a physicianor other qualified health professional and optionally test results (e.g.obtained for biomarkers from samples taken from the subject) and/orhealth changes and treatment endpoints are monitored by a healthprofessional. In some embodiments, the pharmaceutical glycan therapeuticcomposition is administered by a health professional.

In one embodiment, a subject in need thereof can undergo repeatedcourses of treatment with a pharmaceutical glycan therapeuticcomposition. The course of treatment can be repeated when symptomsreappear or increase to an undesirable level. Alternatively, the courseof treatment can be repeated at regular or predetermined intervals.Thus, treatment can be repeated after about one month, two months, threemonths, four months, six months, eight months, ten months, one year, 18months, two years, three years, four years, five years, or more thanfive years, or any combination thereof (e.g., treatment can be repeatedafter one year, then every two to five years thereafter). The treatmentcan be repeated in the same form (e.g., duration, dosage, timing ofdosage, additional substances, etc.) as used in the first treatment orit can be modified. For example, treatment duration can be shortened orlengthened, dosage can be increased or decreased. Optionally, treatmentwith the glycan therapeutic can occur in combination with a differentnumber or compositions of agents, e.g., containing more or less of othersubstances, or fewer or more substances (such as, e.g., a prebioticsubstance, a probiotic bacterium or a therapeutic agent) in addition tothe glycan therapeutic.

Additional substances can be given in conjunction with a pharmaceuticalglycan therapeutic composition. These substances can enhance the actionof the doses of glycan therapeutic by, e.g., encouraging the growth ofbacteria in the GI tract that alleviate symptoms of the gastrointestinaldisease, disorder or condition, increasing adhesion of probiotic orbeneficial commensal bacteria in the niche or in the gut. Thesesubstances can be given prior to treatment with glycan therapeutic,during treatment with glycan therapeutic, after treatment with glycantherapeutic, or any combination thereof. If administered during glycantherapeutic treatment, they can be administered with the dose of glycantherapeutic being given, or before or after the dose of glycantherapeutic, or any combination thereof. In one embodiment substances ofuse in conjunction with a pharmaceutical glycan therapeutic compositioninclude a probiotic microbe(s), prebiotics, therapeutic agents, orbuffers/carriers/excipients. One or more of these substances can be usedin combination with pharmaceutical glycan therapeutic composition at anysuitable time before, during, after treatment, or some combinationthereof.

Definitions

“Abundance” of a microbial taxa as used herein is a relative term andrefers to the relative presence of a microbial taxa to other taxa in acommunity in a defined microbial niche, such as the GI tract, or in theentire host organism (e.g. a human or a laboratory animal model ofdisease).

“Acquire” or “acquiring” as the terms are used herein, refer toobtaining possession of a value, e.g., a numerical value, or image, or aphysical entity (e.g., a sample), by “directly acquiring” or “indirectlyacquiring” the value or physical entity. “Directly acquiring” meansperforming a process (e.g., performing a synthetic or analytical methodor protocol) to obtain the value or physical entity. “Indirectlyacquiring” refers to receiving the value or physical entity from anotherparty or source (e.g., a third party laboratory that directly acquiredthe physical entity or value). Directly acquiring a value or physicalentity includes performing a process that includes a physical change ina physical substance or the use of a machine or device. Examples ofdirectly acquiring a value include obtaining a sample from a humansubject. Directly acquiring a value includes performing a process thatuses a machine or device, e.g., an NMR spectrometer to obtain an NMRspectrum.

The “colonization” of a host organism includes the non-transitoryresidence of a bacterium or other microbial organism. As used herein,“reducing colonization” of a host subject's microbiota, such as in theGI tract by a pathogenic bacterial taxa includes a reduction in theresidence time of the pathogenic bacterial taxa in the niche as well asa reduction in the number, concentration or abundance of the pathogenicbacterial taxa in the niche or adhered to the surface of the niche.Measuring reductions of adherent pathogenic bacterial taxa may bedemonstrated, e.g., by a biopsy sample, or reductions may be measuredindirectly, e.g., by measuring the pathogenic burden, e.g., in the GItract of a host.

“Distinct” as used herein, e.g. with reference to a species in a glycantherapeutic, is meant to denote that it is chemically and/orstructurally different from another. For example, two sugars are“distinct” if they are chemically different, e.g. a fucose and a xylose,or structurally different, e.g. cyclic vs. acyclic, L- vs. D-form. Twodimers are distinct if they consist of the same two monomers but onepair contains alpha-1,4 bond and the other contains a beta-1,6 bond.Distinct entities may have any other suitable distinguishingcharacteristic or property that can be detected by methods known in theart and/or described herein.

“Diversity of a microbial community” or “microbial diversity” as usedherein refers to the diversity found in the microbiota of a given nicheor within a host subject. It can relate to the number of distinctmicrobial taxa and/or richness within the host or niche. Diversity canbe expressed, e.g. using the Shannon Diversity index (Shannon entropy),alpha-beta diversity, total number of observed OTUs, or Chao1 index, asdescribed herein. In some embodiments, the glycan therapeutics describedherein modulate (e.g. increase or decrease) diversity within a microbialcommunity, which may be expressed using Shannon entropy as a measure.For example, the more unequal the abundances of the bacterial taxa, thelarger the weighted geometric mean of the p_(i) values in Shannon'sformula, and the smaller the corresponding Shannon entropy. Ifpractically all abundance is concentrated to one taxa, and the othertaxa are very rare (even if there are many of them), Shannon entropyapproaches zero. When there is only one taxa Shannon entropy exactlyequals zero.

As used herein, a “dosage regimen”, “dosing regimen”, or “treatmentregimen” is a modality of drug administration that achieves atherapeutic objective. A dosage regimen includes definition of one, two,three, or four of: a route of administration, a unit dose, a frequencyof dosage, and a length of treatment.

“Dysbiosis of the gastrointestinal microbiota” refers to an imbalancedstate of the microbiota, e.g., within the GI tract, in which the normaldiversity, proportion of a first bacterial taxa to a second bacterialtaxa and/or function of the ecological network is disrupted ordisturbed. This undesired, e.g., unhealthy, state can be due to a numberof factors including, but not limited to, a decrease or increase in thediversity of the microbiota (e.g. bacterial taxa), the overgrowth of oneor more pathogens or pathobionts, or the shift to an ecologicalmicrobial community that no longer provides an essential function to thehost subject, and, in an embodiment, therefore no longer promotes healthor, which is associated with unwanted symptoms in the subject.

“Ecological Niche” or simply “Niche” refers to the ecological space inwhich an organism or group of organisms occupies (such as the GI tractor one or more subsection of the GI tract, such as, e.g., the stomach,the large or small intestine, the rectum, etc.). In some embodiments,niche specifically refers to a space that microorganisms occupy. Nichemay describe how an organism or population of organisms responds to thedistribution of resources, physical parameters (e.g., host tissue space)and competitors (e.g., by growing when resources are abundant, and whenpredators, parasites and pathogens are scarce) and how it in turn altersthose same factors (e.g., limiting access to resources by otherorganisms, acting as a food source for predators and a consumer ofprey).

By the terms “effective amount” and “therapeutically effective amount”of a pharmaceutical composition or a drug agent is meant a sufficientamount of the composition or agent to provide the desired effect. Insome embodiments, a physician or other health professional decides theappropriate amount and dosage regimen. An effective amount also refersto an amount of a pharmaceutical composition or a drug agent thatprevents the development or relapse of a medical condition.

As used herein, a “glycan therapeutic preparation” (also referred to asa “preparation of glycan therapeutics”, “glycan preparation” or “glycantherapeutic”) is a preparation comprising glycans (sometimes referred toas glycan species) that exhibits a therapeutic effect. A glycantherapeutic comprises a synthetic mixture of a plurality of mono-, di-,oligomeric and/or polymeric glycan species (e.g. oligo- and/orpolysaccharides, sometimes referred to as “oligosaccharides”), whereinthe oligomeric and/or polymeric glycan species comprise glycan unitsthat are linked by glycosidic bonds. A glycan therapeutic may beformulated into a pharmaceutical composition or medical food for humanuse. A glycan therapeutic may be formulated in any suitable dosage formincluding a kit. In some embodiments, preparations of glycantherapeutics do not contain one or more naturally occurring oligo- orpolysaccharide, including: glucooligosaccharide, mannanoligosaccharide,inulin, lychnose, maltotretraose, nigerotetraose, nystose, sesemose,stachyose, isomaltotriose, nigerotriose, maltotriose, melezitose,maltotriulose, raffinose, kestose, fructooligosaccharide,2′-fucosyllactose, galactooligosaccharide, glycosyl, idraparinux,isomaltooligosaccharide, maltodextrin, xylooligosaccharide, agar,agarose, alginic acid, alguronic acid, alpha glucan, amylopectin,amylose, arabioxylan, beta-glucan, callose, capsulan, carrageenan,cellodextrin, cellulin, cellulose, chitin, chitin nanofibril,chitin-glucan complex, chitosan, chrysolaminarin, curdlan, cyclodextrin,alpha-cylcodextrin, dextran, dextrin, dialdehyde starch, ficoll,fructan, fucoidan, galactoglucomannan, galactomannan,galactosamineogalactan, gellan gum, glucan, glucomannan,glucoronoxyland, glycocalyx, glycogen, hemicellulose, hypromellose,icodextrin, kefiran, laminarin, lentinan, levan polysaccharide,lichenin, mannan, mucilage, natural gum, paramylon, pectic acid, pectin,pentastarch, phytoglycogen, pleuran, poligeenan, polydextrose,porphyran, pullulan, schizophyllan, sepharose, sinistrin, sizofiran,sugammadex, welan gum, xantham gum, xylan, xyloglucan, zymosan, and thelike. In some embodiments, a glycan exists as a salt, e.g., apharmaceutically acceptable salt.

A “glycan unit” (sometimes referred to as “feed sugar”) as used hereinrefers to the individual unit of a glycan species disclosed herein,e.g., the building blocks from which the glycan species is made. In anembodiment, a glycan unit is a monomer. In an embodiment, a glycan unitis a dimer. In an embodiment a glycan unit is a monosaccharide. In anembodiment, a glycan unit is a disaccharide. In some embodiments, theglycan unit is a carbohydrate and may be selected from a sugar alcohol,a short-chain fatty acid, a sugar acid, an imino sugar, a deoxy sugar,and an amino sugar. In some embodiments, the glycan unit is erythrose,threose, erythulose, arabinose, lyxose, ribose, xylose, ribulose,xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose,talose, fructose, psicose, sorbose, tagatose, fucose, fuculose,rhamnose, mannoheptulose, sedoheptulose, and the like. In someembodiments, the glycan unit is glucose, galactose, arabinose, mannose,fructose, xylose, fucose, or rhamnose. In embodiments, a glycancomprises distinct glycan units, e.g., a first and a secondmonosaccharide, or a first and a second disaccharide, or a monosaccarideand a disaccharide. In embodiments, a glycan comprises distinct glycanunits, e.g., a first, a second, a third, a fourth, and/or a fifthdistinct glycan unit.

As used herein, an “isolated” or “purified” glycan therapeuticpreparation (also sometimes referred to as “polished”) is substantiallypure and free of contaminants, e.g. pathogens or otherwise unwantedbiological material, or toxic or otherwise unwanted organic or inorganiccompounds. In some embodiments, pure or isolated compounds, compositionsor preparations may contain traces of solvents and/or salts (such asless than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, less than 0.5% or0.1% by w/w, w/v, v/v or molar %). Purified compounds are orpreparations contain at least about 60% (by w/w, w/v, v/v or molar %),at least about 75%, at least about 90%, at least about 95%, at leastabout 97%, at least about 98%, or at least about 99% by w/w, w/v, v/v ormolar % the compound(s) of interest. For example, a purified(substantially pure) or isolated preparation of glycan therapeutics isone that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%,99.5%, 99.8%, 99.9% or 100% of the glycan therapeutic by w/w, w/v, v/vor molar % (i.e. not including any solvent, such as e.g. water, in whichthe glycan therapeutic preparation may be dissolved) and separated fromthe components that accompany it, e.g. during manufacture,extraction/purification and/or processing (e.g. such that the glycantherapeutic is substantially free from undesired compounds). Purity maybe measured by any appropriate standard method, for example, by columnchromatography (e.g., size-exclusion chromatography (SEC)), thin layerchromatography (TLC), gas chromatography (GC), high-performance liquidchromatography (HPLC) or nuclear magnetic resonance (NMR) spectroscopy.Purified or purity may also define a degree of sterility that is safefor administration to a human subject, e.g., lacking viable infectiousor toxic agents.

“Microbiome” as used herein refers to the genetic content of thecommunities of microbes that live in and on a subject (e.g. a humansubject), both sustainably and transiently, including eukaryotes,archaea, bacteria, and viruses (including bacterial viruses (e.g.,phage)), wherein “genetic content” includes genomic DNA, RNA such asribosomal RNA and messenger RNA, the epigenome, plasmids, and all othertypes of genetic information. In some embodiments, microbiomespecifically refers to genetic content of the communities ofmicroorganisms in a niche.

“Microbiota” as used herein refers to the community of microorganismsthat occur (sustainably or transiently) in and on a subject (e.g. ahuman subject), including eukaryotes, archaea, bacteria, and viruses(including bacterial viruses, e.g. phage). In some embodiments,microbiota specifically refers to the microbial community in a niche.

“Pathobionts” or “(Opportunistic) Pathogens” as used herein refer tosymbiotic organisms able to cause disease only when certain geneticand/or environmental conditions are present in a subject.

As used herein, the term “pathogenic” (e.g. “pathogenic bacteria”)refers to a substance, microorganism or condition that has thecapability to cause a disease. In certain contexts, pathogens alsoinclude microbes (e.g. bacteria) that are associated with a disease orcondition but for which a (direct) causative relationship has not beenestablished or has yet to be established. As used herein, the term“pathogens” refers to viruses, parasites and bacteria or other pathogensthat may cause infections in a subject, e.g. a human.

As used herein, a “pharmaceutical composition” or “pharmaceuticalpreparation” is a composition or preparation, having pharmacologicalactivity or other direct effect in the mitigation, treatment, orprevention of disease, and/or a finished dosage form or formulationthereof and is for human use. A pharmaceutical composition orpharmaceutical preparation is typically produced under goodmanufacturing practices (GMP) conditions. Pharmaceutical compositions orpreparations may be sterile or non-sterile. If non-sterile, suchpharmaceutical compositions or preparations typically meet themicrobiological specifications and criteria for non-sterilepharmaceutical products as described in the U.S. Pharmacopeia (USP) orEuropean Pharmacopoeia (EP). Pharmaceutical compositions may furthercomprise or may be co-administered with additional active agents, suchas, e.g. additional therapeutic agents. Pharmaceutical compositions mayalso comprise e.g. additional therapeutic agents, polyphenols, prebioticsubstances, probiotic bacteria, pharmaceutically acceptable excipients,solvents, carriers or any combination thereof. “Pharmaceutical glycantherapeutic compositions” (or simply “glycan therapeutic compositions”)are pharmaceutical compositions as described herein comprising glycantherapeutic preparations and optionally additional agents, ingredients,excipients, or carriers. Any glycan therapeutic described herein may beformulated as a pharmaceutical composition.

The term “phenotype” as used herein refers to a set of observablecharacteristics of an individual entity. As example a subject may have aphenotype of “healthy” or “diseased”. Phenotypes describe the state ofan entity and all entities within a phenotype share the same set ofcharacteristics that describe the phenotype. The phenotype of anindividual results in part, or in whole, from the interaction of theentities genome and/or microbiome with the environment.

The term “subject” (in some cases “patient”) as used herein refers toany human subject. The term does not denote a particular age or gender.Subjects may include pregnant women. Subjects may include a newborn (apreterm newborn, a full term newborn), an infant up to one year of age,young children (e.g., 1 yr to 12 yrs), teenagers, (e.g., 13-19 yrs),adults (e.g., 20-64 yrs), and elderly adults (65 yrs and older). Asubject does not include an agricultural animal, e.g., farm animals orlivestock, e.g., cattle, horses, sheep, swine, chickens, etc.

A “substantial decrease” as used herein (e.g. with respect to abiomarker or metabolite) is a decrease of 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.9% or 100%.

A “substantial increase” as used herein (e.g. with respect to abiomarker or metabolite) is an increase of 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%,550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, or morethan 1000%.

“Synthetic” as used herein refers to a man-made compound or preparation,such as a glycan therapeutic preparation, that is not naturallyoccurring. In one embodiment, the polymeric catalyst described herein isused to synthesize the glycans of the preparation under suitablereaction conditions, e.g. by a polymerization reaction that createsoligomers and polymers from individual glycan units that are added tothe reaction. In some embodiments, the polymeric catalyst acts as ahydrolysis agent and can break glycosidic bonds. In other embodiments,the polymer catalyst can form glycosidic bonds. Synthetic glycantherapeutic preparations may also include glycan therapeutics that arenot isolated from a natural oligo- or polysaccharide source. It is to beunderstood that while the glycan therapeutic preparation is not isolatedfrom a natural oligo- or polysaccharide source, the glycan units makingup the glycan therapeutic can be and often are isolated from naturaloligo- or polysaccharide sources, including those listed herein, or aresynthesized de novo.

The terms “treating” and “treatment” as used herein refer to theadministration of an agent or composition to a subject (e.g., asymptomatic subject afflicted with an adverse condition, disorder, ordisease) so as to affect a reduction in severity and/or frequency of asymptom, eliminate a symptom and/or its underlying cause, and/orfacilitate improvement or remediation of damage, and/or preventing anadverse condition, disorder, or disease in an asymptomatic subject whois susceptible to a particular adverse condition, disorder, or disease,or who is suspected of developing or at risk of developing thecondition, disorder, or disease.

EXAMPLES

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only, and are not to beconstrued as limiting the scope or content of the invention in any way.The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins: Structures and Molecular Properties (W.H.Freeman and Company, 1993); Green & Sambrook et al., Molecular Cloning:A Laboratory Manual, 4th Edition (Cold Spring Harbor Laboratory Press,2012); Colowick & Kaplan, Methods in Enzymology (Academic Press);Remington: The Science and Practice of Pharmacy, 22nd Edition(Pharmaceutical Press, 2012); Sundberg & Carey, Advanced OrganicChemistry: Parts A and B, 5th Edition (Springer, 2007).

Example 1 Preparation of Glycan Therapeutics

To a round bottom flask equipped with an overhead stirrer and a jacketedshort-path condenser was added one or more mono- or disaccharides alongwith 3-20% by dry weight of one or more of the catalysts described inU.S. Pat. No. 8,466,242 and WO 2014/031956, which are incorporatedherein by reference in their entirety. Water or another compatiblesolvent (1.54 equiv) was added to the dry mixture and the slurry wascombined at approximately 100 rpm using a paddle sized to match thecontours of the selected round bottom flask as closely as possible. Themixture was then heated to 80-155° C. Once the solids achieved a moltenstate, the vessel was placed under 10-1000 mbar vacuum pressure. Thereaction was stirred for 30 minutes to 8 hours, constantly removingwater from the reaction. Reaction progress was monitored by HPLC. Whensufficient oligomerization had occurred, the stirrer was shut off, thereaction was cooled to room temperature and vented to atmosphericpressure, and the solid mass was dissolved in a volume of watersufficient to create a solution of approximately 50 Brix (grams sugarper 100 g solution). Once dissolution was complete, solid catalyst wasremoved by filtration and the oligomer solution was concentrated toapproximately 50-75 Brix by rotary evaporation. In cases in which anorganic solvent has been used, water immiscible solvents can be removedby biphasic extraction and water miscible solvents can be removed byrotary evaporation concomitant to the concentration step.

Among others, the following 25 glycans were made in multiple batches andtested in various assays described herein:

Single glycan unit (homo-glycans): xyl100, rha100, ara100, gal100,glu100, and man100.

Two glycan units (hetero-glycans): ara50gal50, xyl75gal25, ara80xyl20,ara60xyl40, ara50xyl50, glu80man20, glu60man40, man60glu40, man80glu20,gal75xyl25, glu50gal50, man62glu38, and the hybrid glycans glu90sor10and glu90gly10.

Three glycan units (hetero-glycans): xyl75glu12gal12, xyl33glu33gal33,glu33gal33fuc33, man52glu29gal19, and glu33gal33neu33.

Example 2 Purification of Glycan Therapeutics

Oligo- and polysaccharides synthesized as in Example 1 were dissolved indeionized water to a final concentration of 25-50 Brix. The material wasthen exposed to at least 2 mass equivalents of Dowex Monosphere 88 ionexchange resin. Exposure may occur by swirling in a flask at 120-170 rpmor by filtration through a wet slurry packed column as long as theresidence time is sufficient for the solution to achieve a final pHbetween 3 and 5. The oligomer solution was isolated by filtration (as inthe case of swirled reactions) or elution (as in the case of columnfiltration) and the process was repeated with Dowex Monosphere 77 ionexchange resin in an analogous fashion until the solution pH was above5.5. Finally the solution was exposed to Dowex Optipore SD-2 Adsorbentdecolorizing resin until the solution was sufficiently clarified andfiltered through a 0.2 micron filter to remove residual resin and resinfines. The final solution was then concentrated to 50-85 Brix by rotaryevaporation or to a solid by lyophilization.

Example 3 High-Throughput Preparation of Glycan Therapeutics at SmallScale

The oligomers and polymers typified in Example 1 were synthesized in aparallel fashion in 24-, 48-, or 96-well plates or similarly sizedarrays of 1 dram vials housed in aluminum heating blocks. In thisexample, all liquid transfers were handled by a programmable robot ormanually using calibrated pipettes. To each vial or well was added20-100% by dry weight of one or more of the catalysts described in U.S.Pat. No. 8,466,242 and WO 2014/031956. The plate or heating block wasplaced uncovered in a vacuum oven heated to 50 to 150° C. under a vacuumof 10-800 mbar. The oven vacuum pump was protected by a two-stagecondenser consisting of a recirculating chiller trap followed by a dryice/acetone trap. The plates or blocks are heated for 30 minutes to 6hours under elevated temperature and reduced pressure without stirring.After a pre-established period of time, the oven was vented toatmospheric pressure, the plates or blocks were cooled to roomtemperature, and each well or vial was diluted to approximately 50 Brixwith deionized water. The solid-phase extraction steps described inExample 2 were performed by elution through sequential wet-packedcolumns in which the eluent from each column flows immediately into thetop of the next column at a rate between 2 and 6 bed volumes/hour usinga peristaltic pump or other suitable small pump. The column stack wasthen rinsed with deionized water and the combined effluents areconcentrated by lyophilization to isolate solid powders with residualwater content of 1-10% by mass.

Example 4 Modification of Glycan Therapeutics by Removal of LowMolecular Weight Species

Oligomers or polymers prepared and purified as in Examples 1 and 2 weremodified so as to remove low molecular weight species. The separationwas achieved by osmotic separation. Approximately 45 cm of 1.0 kD MWCOBiotech CE dialysis tubing (31 mm flat width) from Spectrum Labs wasplaced into deionized water and soaked for 10 minutes, then one end wassealed with a dialysis tubing clip. A 25 Brix solution of 8 grams dryoligosaccharide was sterile filtered and sealed into the tube with asecond clip along with a few mL of air to permit the tube to float. Thefilled tube was then placed in a 3 gallon tank of deionized water whichwas stirred with sufficient force to induce slow swirling of the sealedtubes. After 8 hours, the water in the tank was replaced and the tubewas allowed to stir for an additional 16 hours. Once the dialysis wascomplete and the material had a DP2+ yield greater than 95% and a DP3+yield greater than 90%, the dilute solution was sterile filtered andconcentrated in vacuo to a final concentration of approximately 65 Brixor lyophilized to a solid with a residual moisture between 1 and 10%.Alternatively, the separation was achieved by tangential flow filtration(TFF). In this case, 100 mL of 25 Brix glycan sample dissolved indeionized water and sterile filtered was placed into the feed bottle ofa Spectrum Labs KrosFlo Research IIi TFF system that was preparedaccording to the manufacturer's recommendation. The sample was thendiafiltered through a 1 kD mPES MidiKros hollow-fiber filter at atransmembrane pressure of 25 psig. HPLC samples of the feed stock takenevery 0.5 diafiltration volumes were used to determine when the materialhad a DP2+ yield greater than 95% and a DP3+ yield greater than 90% atwhich point the solution was sterile filtered and concentrated in vacuoto a 65 Brix syrup or lyophilized to a solid with residual water contentof 1-10% by mass.

Example 5 Methods for Analyzing Preparations of Glycan Therapeutics

Measurement of Glycan Content by Liquid Refractometry

This experiment was designed to quantitate the amount of glycan in anygiven aqueous solution. A Mettler-Toledo Refracto 30GS portable sugarrefractometer was calibrated using high-purity reverse-osmosis deionizedwater. Several drops of the glycan solution were filtered through a 0.2micron syringe filter directly onto the lens of the refractometer. Themeasurement was taken at room temperature and reported as Brix. Theglycans were routinely concentrated to 75 Brix without obvioussolidification or crystallization at 23° C. Brix can then be convertedto solubility assuming a specific density of water equal to 1.0 g/mL.Thus, 75 Brix (100 grams of solution consisting of 75 grams of glycanand 25 grams of water) equals an aqueous solubility of 3.0 g/mL. As acomparison, the aqueous solubility of D-glucose is reported to be 0.909g/mL (48 Brix) at 25° C. by Sigma-Aldrich.

Monomeric Composition by Hydrolysis and GC-MS

This experiment was designed to quantitate the ratio of monomer contentwithin a given oligosaccharide. Glycosyl composition analysis wasperformed by combined gas chromatography/mass spectrometry (GC/MS) ofthe per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methylglycosides produced from the sample by acidic methanolysis as describedpreviously by Santander et al. (2013) Microbiology 159:1471. Between 100and 200 μg of sample were lyophilized into a suitable test tube.Inositol (20 μg) was added to the sample as an internal standard, thenthe sample was heated to 80° C. in 1M HCl/methanol for 18 hours. Theresulting monosaccharides were then re-acetylated using pyridine andacetic anhydride in MeOH, and per-O-trimethylsilylated with Tri-Sil(Pierce) at 80° C. for 30 minutes. GC/MS analysis of the TMS methylglycosides was performed on an Agilent 7890A GC interfaced to a 5975CMSD, using a Supelco Equity-1 fused silica capillary column (30 m×0.25mm ID). Each peak was assigned to a component sugar based uponcomparison to known standards and integration of the respective peaksallowed clean calculation of the relative percentage of monomers withinan exemplified glycan. In all tested cases, the monomer composition of agiven oligosaccharide matched the input ratio within experimental errorand the output composition matched the input composition within theprecision of the measurement.

Molecular Weight Distribution by Size-Exclusion Chromatography (SEC)

This experiment was designed to quantitate the distribution of molecularweights within a given oligosaccharide. The measurement was made by HPLCusing the method described in Monograph of United States Pharmacopeia,38(6) In-Process Revision: Heparin Sodium (USP37-NF32). Separations wereachieved on an Agilent 1200 HPLC system via a GE superpose 12 columnusing 50 mM ammonium acetate as an eluent at 1.0 mL/min flow rate and anELSD detector. The column temperature was set at 30° C. and dextran (1kD, 5 kD, 10 kD weight) were used to draw a standard curve. A 2 mg/mlsolution of the samples was prepared and passed through a 0.45 μm spinfilter, followed by 40 μl injections into the HPLC. A third-orderpolynomial curve was constructed based on the logarithmic molecularweights and elution volumes of the listed standards. The weight-averagemolecular weight (Mw), the number average molecular weight (Mn), and thepolydispersity index (PDI) for the sample were calculated by comparisonto the standard curve. FIG. 1 shows the curve generated during the SECevaluation of a glu100 sample in which the average molecular weight wasdetermined to be 1212 g/mol or approximately DP7. The upper end ofmolecular weight of the material as defined by the point of the curve at10% of maximum absorption leading the curve was determined to be 4559g/mol or approximately DP28. The lower end of molecular weight of thematerial as defined by 10% of the maximum absorption trailing the curvewas determined to be 200 g/mol or approximately DP1. Similar analysis ofa glu50gal50 sample showed a MW, high mass, and low mass of 1195 g/mol(˜DP7), 4331 g/mol (˜DP27), and 221 g/mol (˜DP1) respectively.

Molecular Weight Distribution by Ion-Affinity Chromatography (IAC)

The proportion of glycan with DP greater than or equal to 2 (DP2+) and 3(DP3+) may be measured by ion-affinity chromatography. A sample ofglycan was diluted out to 50-100 mg/mL and 10 μL of this solution wasinjected onto an Agilent 1260 BioPure HPLC equipped with a 7.8×300 mmBioRad Aminex HPX-42A column and RI detector. Using pure HPLC-gradewater as an eluent, the sample was eluted at 0.6 mL/min through an 80°C. column and an RI detector maintained at 50° C. The peaks representingDP1-6 are assigned by comparison to reference standards and integratedusing the Agilent ChemStation software. Peaks are typically integratedas DP1, DP2, DP3, DP4-7, and DP8+. The DP that is achievable by thereaction described in Example 1 varies from monomer to monomer althoughit is consistent across batches if the procedure is followed correctly,e.g. glucose reliably achieves higher DP values than arabinose. Forexample, across 17 batches of glu100, DP2+ values ranged from 85-93% andDP3+ values ranged from 80-90%. Conversely, across 6 batches of ara100,DP2+ values ranged from 63-78% and DP3+ values ranged from 48-71%.Mixtures of monomers behaved as averages of the individual components.

Alpha-/Beta-Distribution by 2D NMR

This experiment was designed to quantitate the ratio of alpha- andbeta-glycosidic bonds within a given sample by two-dimensional NMR.Approximately 150 mg of 65 Brix oligosaccharide solution was dried tostable mass in a vacuum oven at 45-95° C. under 400 mbar pressure. Thesample was subjected to two cycles of dissolution in D₂O and drying toremove residual H₂O. Once dried, the sample was dissolved in 750 μL D₂Owith 0.1% acetone, placed into a 3 mm NMR tube, and analyzed in a BrukerAvance-III operating at 500.13 MHz 1H (125.77 MHz 13C) equipped with aBruker BBFO probe operating at 21.1° C. The sample was analyzed using aheteroatomic single quantum coherence pulse sequence (HSQC) using thestandard Bruker pulse sequence. Anomeric protons between 4-6 ppm (1H)and 80-120 ppm (13C) were assigned by analogy to glucose as reported inRoslund, et al. (2008) Carbohydrate Res. 343:101-112. Spectra werereferenced to the internal acetone signal: 1H—2.22 ppm; 13C—30.8 ppm.Isomers were quantitated by integration of their respective peaks usingthe MNova software package from Mestrelab Research (Santiago deCompostela, Spain). FIG. 2 shows the anomeric region of a representativespectrum. Table 6 lists the distribution across 13 distinct combinationsof monomers showing the alpha-/beta-ratio to be as high as 4:1 as in thecase of rha100 and as low as 1:1 as in the case of glu50gal50.

TABLE 6 Distribution of alpha- and beta-bonds across batches and typesof glycans glycans alpha-bonds (%) beta-bonds (%) Glu100 58 42 61 39 6040 Gal100 60 40 Glu50gal50 50 50 56 44 Glu33gal33fuc33 55 45 Man100 5743 Man52glu29gal19 76 24 Ara100 67 33 Rha100 80 20 Xyl100 57 43 59 41Xyl75gal25 56 44

Identification of Composition by NMR

This experiment was designed to identify the composition of a glycan by2D-NMR identification of the constituent monomers. Approximately 150 mgof 65 Brix oligosaccharide solution was dried to stable mass in a vacuumoven at 45-95° C. under 400 mbar pressure. The sample was subjected totwo cycles of dissolution in D₂O and drying to remove residual H₂O. Oncedried, the sample was dissolved in 750 μL D₂O with 0.1% acetone, placedinto a 3 mm NMR tube, and analyzed in a Bruker Avance-III operating at500.13 MHz 1H (125.77 MHz 13C) equipped with a Bruker BBFO probeoperating at 70° C. The sample was analyzed using a heteroatomic singlequantum coherence pulse sequence (HSQC) using the standard Bruker pulsesequence. The anomeric region of each glycan spectra derived from asingle sugar monomer was then examined for peaks representing specificglycosidic bonds characteristic to that monomer. Due to thespin-isolated nature of single carbohydrate rings withinpolysaccharides, the HSQC spectra of a glycan with more than one monomeris predicted to be represented by the sum of the HSQC peaks of each ofits constituent sugars. Therefore, each constituent monomer has uniqueHSQC peaks that will appear in any glycan that contains that monomerirrespective of other constituent monomers and furthermore, the monomersused to synthesize a glycan can be determined by identifying thefingerprint peaks unique to each constituent monomer. For example, FIGS.3A-3C show that the HSQC spectra of glu50gal50 is a hybrid of thespectra of glu100 and gal100. Table 7 lists the fingerprint peaks forselected glycan units.

TABLE 7 Diagnostic HSQC peaks for each component sugar. Monomer 1H shift13C shift Glucose 5.42 92.5 5.21 92.8 5.18 93.9 5.08 97.0 5.36 98.4 5.3499.8 5.38 100.3 4.95 98.6 4.62 96.6 4.70 103.6 4.49 103.4 Galactose 5.3792.9 5.24 93.1 5.14 96.0 4.96 99.3 5.31 98.7 5.39 101.4 5.00 101.8 4.80101.3 4.63 97.0 4.56 97.2 4.53 103.1 4.43 104.1 Fucose 5.18 92.9 5.3392.4 5.04 96.3 4.90 99.7 4.52 97.0 4.39 103.6 Mannose 5.37 93.0 5.1694.6 4.88 94.2 5.39 101.7 5.24 101.9 5.13 102.8 5.03 102.7 5.24 105.65.09 108.0 4.88 94.2 4.89 100.0 4.70 101.1 Xylose 5.18 93.0 5.10 94.35.34 98.2 5.31 99.6 5.11 100.8 4.91 99.4 4.56 97.3 4.64 104.2 4.54 103.44.44 102.6 4.44 104.1 Arabinose 5.22 93.2 5.13 93.2 5.29 96.0 5.26 97.25.12 96.6 5.18 99.6 5.06 99.2 4.99 100.0 5.26 101.9 5.06 102.1 4.55 97.44.54 105.2 4.50 105.5 4.38 103.9 Rhamnose 5.21 93.2 5.10 94.5 4.85 94.15.01 95.8 5.35 100.5 5.15 102.2 5.04 102.9 4.78 97.9 4.71 99.0 4.72101.0

At least 5 peaks appeared for each glycan unit used as a startingmaterial in the synthesis of a glycan therapeutic containing 3 or fewerdistinct glycan units. The HSQC spectra of glycan therapeuticscontaining 4 or more distinct glycan units have at least 4 peaks foreach constituent glycan unit.

Branching Analysis

This experiment was designed to quantitate the distribution ofglycosidic regioisomers (branching) within a given oligosaccharide. Forglycosyl linkage analysis, the samples were permethylated,depolymerized, reduced, and acetylated; and the resultant partiallymethylated alditol acetates (PMAAs) analyzed by gas chromatography-massspectrometry (GC-MS) as described by Heiss et al (2009) Carbohydr. Res.344:915. The samples were suspended in 200 μl of dimethyl sulfoxide andleft to stir for 1 day. Permethylation was effected by two rounds oftreatment with sodium hydroxide (15 min) and methyl iodide (45 min). Theaqueous solution was hydrolyzed by addition of 2M trifluoroacetic acidand heating to 121° C. for 2 hours. Solids were isolated in vacuo andacetylated in acetic acid/trifluoroacetic acid. The resulting PMAAs wereanalyzed on an Agilent 7890A GC interfaced to a 5975C MSD (massselective detector, electron impact ionization mode); separation wasperformed on a 30 m Supelco SP-2331 bonded phase fused silica capillarycolumn. FIG. 4A, FIG. 4B, and FIG. 4C show three representative GCspectra from this analysis. These analyses show that the glycans had atleast 0.1-10% of each of the 1,2-; 1,3-; 1,4-, and 1,6-glycoside bondtypes. The materials also contained at least 5% of the branched bondtypes (including but not limited to 1,3,6-; 1,4,6-; or 1,2,4-glycosides)and at least 3% of the monomeric units existed in the furanose form. Aglycan originating from a single monomer consisted of at least 12distinct non-terminal substitution patterns. A glycan originating fromtwo monomers consisted of at least 18 distinct non-terminal substitutionpatterns. A glycan originating from three or more monomers consisted ofat least 24 distinct non-terminal substitution patterns.

Example 6 Collection of Fecal Samples

Fecal samples were collected by providing subjects with the FisherbrandCommode Specimen Collection System (Fisher Scientific) and associatedinstructions for use. Collected samples were stored with ice packs or at−80° C. until processing (McInnes & Cutting, Manual of Procedures forHuman Microbiome Project: Core Microbiome Sampling Protocol A, v12.0,2010, hmpdacc.org/doc/HMP_MOP_Version12_0_072910.pdf). Alternativecollection devices may also be used. For example, samples may becollected into the Globe Scientific Screw Cap Container with Spoon(Fisher Scientific) or the OMNIgene-GUT collection system (DNA Genotek,Inc.), which stabilizes microbial DNA for downstream nucleic acidextraction and analysis. The subjects donating the fecal samples weregiven the manufacturer-supplied instructions for use of each collectiondevice. Aliquots of fecal samples were stored at −80° C. followingstandard protocols known to one skilled in the art.

Example 7 Determining the Titer of Microbial Samples Collected fromFeces and Culturing Samples

To determine the titer of common bacteria of the gastrointestinal tract,fecal samples were collected as described in Example 6 and prepared as a10% weight/volume suspensions in sterile phosphate buffered saline(PBS). Ten-fold serial dilutions were prepared in sterile PBS and plated(100 μL per dilution) to Brucella Blood Agar (Anaerobe Systems;incubated anaerobically to non-selectively titer common member of thegut microbiota, including Bacteroides, or incubated aerobically tonon-selectively titer facultative anaerobes such as Proteobacteria).Bacteroides Bile Esculin Agar (Anaerobe Systems; cultured anaerobicallyto titer Bacteroides fragilis group), Cycloserine-Cefoxitin FructoseAgar (Anaerobe Systems; cultured anaerobically to titer Clostridiumdifficile), Lactobacillus-MRS Agar (Anaerobe Systems; culturedanaerobically to titer Lactobacillus), Eosin Methylene Blue Agar(Teknova; cultured aerobically to titer Escherichia coli and otherGram-negative enteric bacteria), Bile Esculin Agar (BD; culturedaerobically to titer Enterococcus species), Bifidobacterium SelectiveAgar (Anaerobe Systems; to titer Bifidobacterium species), or MacConkeyAgar (Fisher Scientific; to titer E. coli and other Gram-negativeenteric bacteria) may also be used. Plates were incubated at 37° C.under aerobic or anaerobic conditions as appropriate for the targetspecies. After 24-48 hours, colonies were counted and used toback-calculate the concentration of viable cells in the original sample.

To non-selectively culture samples containing bacteria collected from ahuman or laboratory animal model, rich media or agar such as BrucellaBlood Agar (Anaerobe Systems), Brain Heart Infusion Broth (Teknova), orChopped Meat Glucose Broth (Anaerobe Systems) were used. A minimal mediaformulation such as M9 (Life Technologies) supplemented with aminoacids, carbon sources, or other nutrients as needed were used tonon-selectively culture bacteria during in vitro assays testing theeffects of glycans or other compounds on bacterial populations.Alternatively, other minimal media formulations known to one skilled inthe art were used, for example, as reported in Martens et al. (MucosalGlycan Foraging Enhances Fitness and Transmission of a SaccharolyticHuman Gut Bacterial Symbiont, 2008, Cell Host & Microbe, 4:447-457).Alternatively, fecal slurries at a concentration of 0.1%-10%weight/volume in PBS were used in the presence or absence of additionalmedia elements for in vitro assays testing the effects of glycans orother compounds on bacterial populations.

Example 8 Single Strain Growth Assays

An in vitro assay was performed to assess the ability of variousbacterial strains, including commensals and pathogens of thegastrointestinal tract, to utilize different glycans as growthsubstrates. This assay was designed to assess the ability of selectedglycans to promote the growth of healthy-state microbiota. Additionally,the ability of selected glycans to promote the growth of commensals wascompared to the ability of the glycans to promote the growth of microbesassociated with a disease state. By testing preparations of glycansagainst a panel of bacteria (individually) which are characteristic of ahealthy or disease state preparations of glycans that selectivelyenhance the growth of healthy-state bacteria over disease-state bacteriacan be selected. Bacterial strains were handled at all steps in ananaerobic chamber (AS-580, Anaerobe Systems) featuring a palladiumcatalyst. The chamber was initially made anaerobic by purging with ananaerobic gas mixture of 5% hydrogen, 5% carbon dioxide and 90% nitrogenand subsequently maintained in an anaerobic state using this sameanaerobic gas mixture. Anaerobicity of the chamber was confirmed dailyusing Oxoid anaerobic indicator strips that change color in the presenceof oxygen. All culture media, assay plates, other reagents and plasticconsumables were pre-reduced in the anaerobic chamber for 24-48 hoursprior to contact with bacteria. Glycans ara50gal50, glu33gal33fuc33,glu50gal50, gal100, glu100, ara50xyl50, xyl100, ara100, ara60xyl40,rha100, gal75xyl25, glu90gly10, man62glu38, man52glu29gal19, and twocommercially available controls: acacia fiber (Acacia Fiber OrganicPowder; NOW Foods, Bloomingdale Ill.) and FOS (Nutraflora FOS; NOWFoods, Bloomingdale Ill.) were prepared at 5% w/v in water,filter-sterilized and added to Costar 3370 assay plates for a finalconcentration of 0.5% w/v in the assay, with each glycan assayed in twonon-adjacent wells and dextrose and water supplied as positive andnegative controls.

Bacterial isolates were obtained from the American Type CultureCollection (ATCC) and Leibniz Institute DSMZ-German Institute ofMicroorganisms and Cell Cultures (DSMZ). 8 commensal species(Bacteroides caccae ATCC 43185 “BCA.26”, Prevotella copri DSM 18205“PCO.72”, Bacteroides thetaiotaomicron ATCC 29741 “BTH.8”, Bacteroidescellulosilyticus DSM 14838 “BCE.71”, Clostridium scindens ATCC 35704“CSC.32”, Ruminococcus obeum ATCC 29714 “ROB.74”, Clostridium nexileATCC 27757 “CNE.31” and Parabacteroides distasonis ATCC 8503 “PDI.6”)and three pathogenic species (Clostridium difficile ATCC BAA-1382“CDI.23” and ATCC 43255 “CDI.24”, Enterococcus faecium ATCC 700221“EFM.66” and Salmonella enterica ATCC 27869 “SEN.52”) were grownanaerobically on Brucella Blood Agar (Anaerobe Systems), a pre-reducedenriched medium including enzymatic digests of casein and animaltissues, yeast extract, sodium chloride, dextrose, sodium bisulfite,sheep's blood, hemin and Vitamin K1, for 18-48 hours at 37° C. Thecommensal species Akkermansia muciniphila ATCC BAA-835 “AMU.73” wasgrown anaerobically on MTGE agar plates (Anaerobe Systems), a richmedium including a protein formulation, yeast extract, vitamin K1 andvolatile fatty acids. Inocula were prepared by scraping colonies fromagar plates, suspending them in phosphate buffer, determining the cellsuspensions' optical density at 600 nM (OD₆₀₀) in a Costar 3370polystyrene 96-well flat-bottom assay plate using a Biotek Synergy 2plate reader with Gen5 2.0 All-In-One Microplate Reader Softwareaccording to manufacturer's protocol, and diluting the cells to OD₆₀₀0.01-0.02 final in defined and semi-defined media that were preparedwithout sugars. D. formicigenerans, P. distasonis, C. difficile and E.faecium isolates were tested in 900 mg/L sodium chloride, 26 mg/Lcalcium chloride dihydrate, 20 mg/L magnesium chloride hexahydrate, 10mg/L manganese chloride tetrahydrate, 40 mg/L ammonium sulfate, 4 mg/Liron sulfate heptahydrate, 1 mg/L cobalt chloride hexahydrate, 300 mg/Lpotassium phosphate dibasic, 1.5 g/L sodium phosphate dibasic, 5 g/Lsodium bicarbonate, 0.125 mg/L biotin, 1 mg/L pyridoxine, 1 m/Lpantothenate, 75 mg/L histidine, 75 mg/L glycine, 75 mg/L tryptophan,150 mg/L arginine, 150 mg/L methionine, 150 mg/L threonine, 225 mg/Lvaline, 225 mg/L isoleucine, 300 mg/L leucine, 400 mg/L cysteine, and450 mg/L proline (Theriot C M et al. Nat Commun. 2014; 5:3114),supplemented with 3.5% (v/v) Chopped Meat Glucose Broth (CMG, AnaerobeSystems), a rich medium including yeast extract, peptone, chopped beefand phosphate buffer. B. thetaiotaomicron, B. caccae, B.cellulosyliticus and S. enterica were tested in 100 mM potassiumphosphate buffer (pH 7.2), 15 mM sodium chloride, 8.5 mM ammoniumsulfate, 4 mM L-cysteine, 1.9 μM hematin, 200 μM L-histidine, 100 μMmagnesium chloride, 1.4 μM iron sulfate heptahydrate, 50 μM calciumchloride, 1 μg/mL vitamin K3 and 5 ng/mL vitamin B12 (Martens E C et al.Cell Host & Microbe 2008; 4, 447-457). C. scindens, P. copri and R.obeum were tested in 10 g/L tryptone peptone, 5 g/L yeast extract, 0.5g/L L-cysteine hydrochloride, 0.1 M potassium phosphate buffer pH 7.2, 1μg/mL vitamin K3, 0.08% w/v calcium chloride, 0.4 μg/mL iron sulfateheptahydrate, 1 μg/mL resazurin, 1.2 μg/mL hematin, 0.2 mM histidine,0.05% Tween 80, 0.5% meat extract (Sigma), 1% trace mineral supplement(ATCC), 1% vitamin supplement (ATCC), 0.017% v/v acetic acid, 0.001% v/visovaleric acid, 0.2% v/v propionic acid and 0.2% v/v N-butyric acid(Romano K A et al. mBio 2015; 6(2):e02481-14); for C. nexile and A.muciniphila, this medium was supplemented with 3.5% v/v final of CMGbroth. Bacteria were exposed to glycans ara50gal50, glu33gal33fuc33,glu50gal50, gal100, glu100, ara50xyl50, xyl100, ara100, ara60xyl40,rha100, gal75xyl25, man62glu38, man52glu29gal19, commercial acaciafiber, commercial FOS and dextrose at a final concentration of 0.5% w/vin 96-well microplates, 200 μL final volume per well, at 37° C. for18-48 hours, anaerobically, until turbidity was observed in the positivegrowth control wells containing 0.5% w/v dextrose. OD₆₀₀ measurementsfor each isolate at the end of the incubation period were obtained usinga Biotek Synergy2 reader with Gen5 2.0 software according tomanufacturer's specifications. Measurements were normalized by dividingthe OD₆₀₀ readings of the isolate on test glycans by the OD₆₀₀ of theisolate in medium supplemented with 0.5% w/v dextrose to facilitatecomparison of glycan utilization by strains that grow withinsignificantly different OD₆₀₀ ranges. Tables 8 and 9 summarize theresults obtained.

Key to glycans glycan # glycan identity % DP3+ 1 gal75xyl25 50 2glu50gal50 68-80 3 gal100 78-83 4 glu33gal33fuc33 54-82 5 man62glu38 636 ara50gal50 73 7 glu100 79-80 8 man52glu29gal19 47-77 9 xyl100 37-73 10ara50xyl50 63 11 ara100 48-85 12 ara60xyl40 70 13 rha100 45-49 14 FOS 15acacia fiber

Most glycans supported growth of the commensal strains tested in thisassay. Gal75xyl25, glu33gal33fuc33, glu50gal50, gal100, man62glu38,ara50gal50, glu100, man52glu29gal19, ara50xyl50 xyl100 and ara100supported growth of at least 5 of 9 commensals (see Table 8).

TABLE 8 Glycan-supported growth of commensal bacteria. IsolatesCommensals, Average Normalized Growth (NG) glycan # w/NG >0.2 PDI.6BTH.8 BCA.26 CNE.31 CSC.32 BCE.71 PCO.72 AMU.73 ROB.74 1 9/9 +++ ++++ + + ++ +++ ++ + 2 9/9 +++ + ++ + + ++ +++ + + 3 9/9 +++ + + ++ + +++ + + 4 9/9 +++ + + + + + ++ + + 5 8/9 +++ ++ ++ + + ++ − + + 6 8/9+++ + + − + + ++ + + 7 8/9 +++ + + + − + ++ + + 8 7/9 +++ + + − + +− + + 9 6/9 +++ + + − − + ++ − + 10 6/9 ++ + + − − + ++ − + 11 5/9 − + +− − + ++ − + 12 4/9 ++ − + − − + + − − 13 2/9 − − − − − − + − + 14 5/9+++ ++ ++ − − − ++ − ++ 15 1/9 + − − − − − − − − Key to Symbols SymbolNG − <0.2 + 0.2-6 ++ 0.6-1.2 +++ >1.2

Some glycans supported growth of tested commensals better than testedpathogens: ara50gal50, glu33gal33fuc33, gal100, glu100 ara50xyl50,xyl100 and commercial FOS control supported growth of 5 or more of 9commensal isolates and 2 or fewer of 4 pathogenic isolates, withnormalized growth values of at least 0.2. In the assay, glu50gal50 andgal75xyl25 each supported the majority of commensals and pathogens withnormalized growth values of at least 0.2; however, they supported ahigher level of growth with a larger fraction of commensals thanpathogens: gal75xyl25 produced normalized growth values >0.6 for 6 of 9commensals and 1 of 4 pathogens, and glu50gal50 produced normalizedgrowth values >0.6 for 4 of 9 commensals and 0 of 4 pathogens. In theassay, one glycan supported growth of pathogens as well as commensals:man62glu38 supported growth of 4 of 4 pathogens and at least 8 of 9commensals with normalized growth values of at least 0.2 and 3 of 4pathogens and 6 or fewer of 9 commensals with normalized growthvalues >0.6. In the assay, one glycan did not support a majority ofcommensals or pathogens: rha100 and commercial acacia fiber controlsupported 2 or fewer of 9 commensals and 2 or fewer of 4 pathogens withnormalized growth values of >0.2 (see Table 9).

TABLE 9 Differential growth of commensals and pathogenic bacteria onselected glycans glycan Commensals Pathogens Commensals # w/NG >0.2w/NG >0.2 PDI.6 BTH.8 BCA.26 CNE.31 CSC.32 3 9/9 0/4 +++ + + ++ + 7 8/92/4 +++ + + + − 6 8/9 0/4 +++ + + − + 10 6/9 0/4 ++ + + − − 9 6/9 0/4+++ + + − − 14 5/9 1/4 +++ ++ ++ − − 1 9/9 3/4 +++ ++ ++ + + 2 9/9 4/4+++ + ++ + + 5 8/9 4/4 +++ ++ ++ + + 13 2/9 0/4 − − − − − 15 1/9 2/4 + −− − − glycan Commensals Pathogens # BCE.71 PCO.72 AMU.73 ROB.74 CDI.23CDI.24 SEN.52 EFM.66 3 + ++ + + − − − − 7 + ++ + + + − − + 6 + ++ + + −− − − 10 + ++ − + − − − − 9 + ++ − + − − − − 14 − ++ − ++ − − − + 1 +++++ ++ + + − + + 2 ++ +++ + + + + + ++ 5 ++ − + + ++ ++ + ++ 13 − + − +− − − − 15 − − − − + − − + Key to Symbols Symbol NG − <0.2 + 0.2-6 ++0.6-1.2 +++ >1.2

These data suggest that glycan therapeutics support growth of commensalbacteria and certain sub-groups of glycans differentially support growthof commensals over pathogens.

Example 9 Effect of Glycans on Microbial Populations In Vitro

To determine the desired composition of glycans, bacterial cultures aregrown in the presence of candidate glycans and assayed for their growth,community composition (e.g., by 16S rRNA gene sequencing), production ofmetabolites, and phenotypic or transcriptomic properties. Desiredglycans are selected based on their ability to elicit desired propertieswithin the bacterial culture. Bacterial cultures include monocultures,mixed cultures, cultures isolated from humans or laboratory animalmodels, cultures isolated from a human or laboratory animal model andspiked with an isolate or collection of isolates, or cultures isolatedfrom a human or laboratory animal model and depleted of a collection ofspecies (for example, by application of an antibiotic). The titer of thebacterial cultures is determined as in Example 7 and the composition andproperties of the bacterial cultures are quantified as described hereinor using standard protocols. This assay can be performed in the presenceof antibiotics or other test compounds. The results obtained from the invitro assays are compared with those obtained after treating humans withglycans or administering the glycans to a laboratory animal in an animalmodel as described, e.g., in Example 10 and Example 12, thusestablishing the in vitro—in vivo correlation of results.

Example 10 Effect of Glycans on the Intestinal Microbiota of Naïve Mice

This experiment was carried out to assess the effect of glycantherapeutics on the gut microbiota and short term weight of naïve mice.In this model, normal mice are administered glycans in their drinkingwater over a period of 6 days with fecal samples taken from each mousefor 16S rRNA analysis.

Mice, C57Bl/6 (B6N Tac), mouse pathogen free (MPF; Taconic Biosciences,Germantown, N.Y.) aged 8-10 weeks were housed singly in cages, with 6animals per dose group. Animals were fed PicoLab Rodent Diet 20 (“5053”;LabDiet, St. Louis, Mo.) or zero fiber diet (“ZFD”; Modified rodent dietAIN-93G: D15091701, Research Diets, New Brunswick, N.J.) ad libitumthroughout the course of the study and had free access to water. Micewere maintained on a 12 h light/dark cycle. Mice were acclimated for 7days (days −7 to −1) prior to glycan administration.

Glycans were administered to the mice by inclusion in their drinkingwater at 1% weight/volume (w/v) from day 0 through day 5. Control micereceived water containing no glycan. Fresh fecal collections wereperformed for each mouse from days −2 to 5. Mouse weights were monitoredon days −1, 1, 3 and 4. Body weights of the mice did not changesignificantly throughout the course of the study.

Genomic DNA was extracted from the fecal samples and variable region 4of the 16S rRNA gene was amplified and sequenced (Earth MicrobiomeProject protocol www.earthmicrobiome.org/emp-standard-protocols/16s/ andCaporaso J G et al. 2012. Ultra-high-throughput microbial communityanalysis on the Illumina HiSeq and MiSeq platforms. ISME J.).Operational Taxonomic Units (OTUs) were generated by aligning 16S rRNAsequences at 97% identity. Microbial communities were compared to eachother using UniFrac distance metric (Lozupone C. et al., Appl. Environ.Microbiol. December 2005 vol. 71 no. 12 8228-8235).

Significant changes were observed when mice were administered a xyl 100preparation. UniFrac distances between microbiota sampled at one daybefore and 5 days after glycan administration were significantly largerin mice treated with xylose compared to mice who did not receive anyglycan (p=0.0043, Mann-Whitney test, FIG. 7). Alpha diversity wasmeasured by calculated Shannon Index in microbiota before and afterglycan or water administration. Shannon index significantly decreasedafter 5 days of xylose administration (p=0.0313, Wilcoxon paired test,FIG. 8).

The changes in observed shifts with administration of xylose wereattributed to an increase in relative abundance of sequences assigned togenus Akkermansia (phylum Verrucomicrobia, p=0.0313, Wilcoxon pairedtest, FIG. 9A), and genus Blautia (phylum Firmicutes, familyLachnospiraceae, p=0.0313, Wilcoxon paired test, FIG. 9B).

The most prominent Akkermansia species in the mammalian gut isAkkermansia muciniphila. Its preferred energy source is host intestinalmucin. Consumption of a low fiber diet and high intake of simple sugarsand fat results in decreased mucus production (British Journal ofNutrition/Volume 102/Issue 01/July 2009, pp 117-125, QuantitativeImaging of Gut Microbiota Spatial Organization, Earle K A et al, CellHost Microbe. 2015 Oct. 14; 18(4):478-88). Thinning of intestinal mucusmay result in increased gut permeability and translocation ofmicroorganisms or their components, such as lipopolysaccharide (LPS),which induce inflammation. LPS levels are increased upon consumption ofhigh fat diet in rodents which then develop metabolic syndrome(Metabolic endotoxemia initiates obesity and insulin resistance, Cani PD et al, Diabetes. 2007 July; 56(7):1761-72).

Although Akkermansia muciniphila was not shown to degrade xylose invitro (Example 8, Table 8, AMU.73), other species may be responsible forprimary fermentation of xyl100, such as, e.g., Bacteroidetes, which inturn may induce the growth of Akkermansia. For example, colonization ofgerm free mice with Bacteroides thetaiotaomicron induces mucusproduction by intestinal goblet cells (Wrzosek et al. BMC Biology 201311:). This may create a favorable environment for Akkermansia growth.Consumption of mucus by Akkermansia may stimulate increased mucusproduction and play a role in the restoration of the gut barrier thatprevents leaking of microbial endotoxin LPS. Decreased endotoxemiareduces inflammation and may alleviate symptoms that are associated withmetabolic syndrome. For example, administration of FOS or Akkermansiamuciniphila to rodents in a diet-induced obesity model result indecreased levels of serum LPS and reduced fat mass and body weight.(Cross-talk between Akkermansia muciniphila and intestinal epitheliumcontrols diet-induced obesity, Everard A, PNAS. 2013 May 28;110(22):9066-71).

Akkermansia muciniphila metabolites, including the SCFA propionate, havebeen shown to reduce expression of adiposity regulators Gpr43 andperoxisome proliferator-activated receptor gamma and increase expressionof the gene regulators histone deacetylases Hdac3 and Hdac5 (Lukovac etal. 2014. Differential Modulation by Akkermansia muciniphila andFaecalibacterium prausnitzii of Host Peripheral Lipid Metabolism andHistone Acetylation in Mouse Gut Organoids mBio 5(4):e01438-14). Glycantherapeutics when administered in an effective amount may modulatebacterial species that produce SCFAs and thereby modulate host adiposityand obesity. In the in vitro assay (Example 8), growth of AMU.73, an A.muciniphila isolate, was supported by 8 out of 15 glycans, as shown inTable 8.

Example 11 In Vitro Co-Culture Models to Test the Effect of Glycans onHost Responses to Bacterial Communities at Intestinal Sites

Bacteria can elicit both pro- and anti-inflammatory responses from host(mammalian) cells, and different bacterial species can elicit differenthost responses. Preparations of glycans are used to alter the bacterialpopulation to elicit a desired host response. An in vitro co-culturemodel is used to measure the host responses elicited by bacterialpopulations grown in the presence of glycans. Glycans that promotebacterial populations that elicit beneficial host responses or minimizedetrimental host responses are selected using this assay.

Epithelial cell lines or tissues from the intestine are used in aco-culture model (Haller D, Bode C, Hammes W P, Pfeifer A M A, SchiffrinE J, Blum S, 2000. Non-pathogenic bacteria elicit a differentialcytokine response by intestinal epithelial cell/leucocyte co-cultures.Gut. 47:79-97) (Borruel et al., 2003. Effects of nonpathogenic bacteriaon cytokine secretion by human intestinal mucosa. Am J Gastroenterology.98:865-870). Human enterocyte-like CaCO-2 cells are seeded at a densityof 2.5×10⁵ cells/ml on 25 mm cell culture inserts (0.4 μm nucleoporesize; Becton Dickinson). The inserts are placed into 6-well tissueculture plates (Nunc) and cultured 18-22 days at 37° C./10% CO₂ in DMEM(glutamine, high glucose; Amimed) supplemented with 20% heat-inactivatedfetal calf serum (56° C., 30 minutes; Amimed), 1% MEM non-essentialamino acids (Gibco BRL), 10 μg/ml gentamycin (Gibco BRL), and 0.1%penicillin/streptomycin (10 000 IU/ml/10 000 UG/ml; Gibco BRL). The cellculture medium is changed every second day until the cells are fullydifferentiated. Alternatively, a 3D reconstructed tissue model producedfrom normal, human cell-derived small intestine epithelial andendothelial cells and fibrobasts (Epilntestinal model; MatTekCorporation, Ashland, Mass.) is used. Transepithelial electricalresistance (TEER) is determined using a MultiCell-ERSvoltmeter/ohmmeter. Tissue culture inserts are washed twice withprewarmed antibiotic-free medium prior to challenge with bacterialcultures. Separately, bacterial cultures are grown in the presence ofpreparations of glycan as described in Example 9. After 16-24 hours ofgrowth in the presence of glycans, the bacterial suspensions areprepared in antibiotic-free medium and 10⁶-10⁸ CFU are added toconfluent cell or tissue cultures. The co-cultures are incubated at 37°C. for 4-24 hours.

At the conclusion of the co-incubation period, the supernatant iscollected and analyzed for inflammatory and immunomodulatory cytokinesincluding IL-1α, IL-1β, TNF, IL-8, RANTES, IL-10, TGF-β, IFN-γ, IL-4,IL-6, IL-12, IL-17, and IL-23. This analysis is performed by enzymelinked immunosorbent assay (ELISA) or other comparable quantificationtechnique (e.g., Luminex Assay; Life Technologies, Carlsbad, Calif.)following standard protocols. To analyze a broader range of responses,gene expression (e.g., by microarray) or transcriptomic (e.g., byRNA-Seq) analysis is performed by lysing the cells, purifying RNA, andfollowing standard protocols. This procedure is used to analyze theexpression of genes encoding inflammatory cytokines, immunomodulatorycytokines, antimicrobial peptides, and other relevant host responses.

Example 12 Effect of Glycans in a Mouse Model of Clostridium difficileInfection

This experiment was conducted to analyze the effects of glycantherapeutics in a mouse model of Clostridium difficile infection (Chenet al, 2008, A Mouse Model of Clostridium difficile-Associated Disease.Gastroenterology 135(6), 1984-1992). In this model, normal mice wereexposed to an antibiotic regimen that renders them susceptible to C.difficile infection and disease symptoms, similar with respect to theirdevelopment and manifestation of human clinical disease. In humans, thedisease is most often the result of exposure to broad spectrumantibiotics which is thought to result in intestinal dysbiosis andsubsequent increased colonic colonization with C. difficile. Theincreased bio-burden of C. difficile leads to toxin production by thebacterium and colonic inflammation. Clinical manifestations in humansinclude diarrhea, weight loss, intestinal inflammation, fever anddehydration. The clinical incidence of C. difficile infection anddisease is about 750,000 cases per year in the US.

Mice (female C57BL/6, 8-10 weeks old, 16-18 grams each; HarlanLaboratories, Indianapolis, Ind.) were housed in groups of 3 per cage,with 4 cages per treatment group. Mice were exposed to a cocktail ofantibiotics in their drinking water for a period of 9 days, starting onday −14, fourteen days before Clostridium difficile challenge on day 0.The antibiotic cocktail consisted of 1% glucose, kanamycin (0.5 mg/ml),gentamicin (0.044 mg/ml), colistin (1062.5 U/ml), metronidazole (0.269mg/ml), ciprofloxacin (0.156 mg/ml), ampicillin (0.1 mg/ml) andvancomycin (0.056 mg/ml). Three days prior to Clostridium difficilechallenge (on Day −3), mice received a single dose of clindamycin (10mg/kg) in a volume of 0.5 mL distilled water by oral gavage (PO) (seeTable 10). All chemicals were purchased from Sigma-Aldrich Corp. (St.Louis, Mo.). On day 0, mice were challenged with Clostridium difficile(strain VPI 10463 (ATCC-43255)) spores at approximately 4.5 log₁₀ sporesper mouse, PO, in a dose volume of 0.5 mL distilled water.

TABLE 10 Treatments Treatment Route of administration Treatment durationPlain water (control) Drinking water QD days −15 to 6 (L) Vancomycin(control) PO gavage QD days 0 to 4 glu100 (L) Drinking water QD days −15to 6 glu100 (S) Drinking water QD days −1 to 6 (S) glu33gal33fuc33 (L)Drinking water QD days −15 to 6 glu33gal33fuc33 (S) Drinking water QDdays −1 to 6 ara100 (L) Drinking water QD days −15 to 6 ara100 (S)Drinking water QD days −1 to 6 glu50gal50 (L) Drinking water QD days −15to 6 glu50gal50 (S) Drinking water QD days −1 to 6 FOS**FOS (NutrafloraFOS; Drinking water QD days −15 to 6 NOW Foods, Bloomingdale IL) (L) FOS(S) Drinking water QD days −1 to 6

A. Disease-Associated Phenotypes

Disease-associated phenotypes were recorded for a duration of ten daysstarting on the day of Clostridium difficile challenge (on day 0)through day 10. The phenotypes, including lethargy, hunched posture andruffled coat, wet tail/abdomen and hypothermia were scored from 0 to 4:Normal=0; Lethargic=1; Lethargic+Hunched=2; Lethargic+Hunched+Wettail/abdomen (diarrhea)=3; and Lethargic+Hunched+Wettail/abdomen+Hypothermic=4. Death (day 0 to day 10 or 11) and weight(day 1 to day 7 and day 10) of the animals was also monitored andrecorded. Mice that showed a body weight loss of more than 25% relativeto Day 0 were humanely euthanized.

In these studies, all animals with wet tail/abdomen (diarrhea; clinicalscore of “3”) progressed to death. Diarrhea is an indication of colonicinflammation and hemorrhage and is a contributing factor to the weightloss seen in rodent models of C. difficile colonization/infection.

A treatment's ability to protect animals from death through day 6 inthese experiments indicates disease prevention, e.g., in a human. Atreatment's ability to protect animals from death between days 7 and 11indicates that animals were protected from disease relapse in thesemodels.

In addition, carriage of Clostridium difficile spores, which isindicative of Clostridium difficile gut colonization in both mice andhumans, was assessed daily from day 0 to day 6. For spore CFUenumeration, fecal pellets from the mice were suspended in 50% ethanolin phosphate-buffered saline (PBS) and vortexed. Samples were incubatedat room temperature for 1 hour, vortexed well and serially-diluted inPBS. The resulting suspensions were applied to Clostridiumdifficile-selective TCCFA agar (Teknova, Hollister Calif.) and grown inan anaerobic atmosphere at 37° C. overnight to enumerate CFU.

Effects of treatments was assessed by comparing the disease-associatedphenotypes in glycan-treated, untreated (plain water) andvancomycin-treated groups. FOS is a commercially-available,non-digestible fructooligosaccharide. Clinical trials using commercialFOS as an intervention for antibiotic and Clostridiumdifficile-associated diarrhea (Lewis et al., Failure of dietaryoligofructose to prevent antibiotic-associated diarrhea. AlimentPharmacol Ther 2005; 21: 469-477) and Clostridium difficile relapse(Lewis et al, Effect of the Prebiotic Oligofructose on Relapse ofClostridium difficile-Associated Diarrhea: A Randomized, ControlledStudy. Clin Gastroent Hepatol 2005 (3):442-448.) have yielded differentresults, with the former trial showing no effect of FOS treatment andthe latter demonstrating a reduction in disease recurrence.

Survival

With regard to animal survival, there was a significant differencebetween animals treated with water (vehicle) versus animals treated witheither glycans or vancomycin (FIG. 10). Animal treatments werediscontinued on day 4 (for vancomycin) and day 6 (for glycans) andanimal deaths/clinical scores assessed through day 10 or 11. For watertreated animals, 75% of the animals died by day 4 (FIG. 10). All (100%)of vancomycin-treated animals survived to day 7. However, 17% of theanimals in the vancomycin-treated group died by day 10. The remainingsurvivors in the vancomycin-treated group had clinical scores above 0.For glycan-treated groups (L=treatment days −15 to 6; S=days −1 to 6),deaths through day 6 were as follows: For glu100 (L), glu33gal33fuc33(L), and glu50gal50 (L and S) death was 0%. For ara100 (S),glu33gal33fuc33 (S), glu100 (L), and FOS (L) 8% of animals died. Forara100 (L), and FOS (S) 25% of the animals died. All glycan-treatedsurvivors remained at a clinical score of 0 and there were no deathsfrom day 7-11. No animals in the glu100 (L and S), glu33gal33fuc33 ((Land S), ara100 (S) and glu50gal50 (L and S) groups exhibited a clinicalscore of “3”, indicating that these treatments prevented diarrhea. Twoof the animals in the untreated control group exhibited diarrhea.

Weight Loss

Weight loss in animals treated with any of the glycans tested wassignificantly less than water-treated animals (FIG. 11, ***P<0.001;repeated measures ANOVA, Bonferonni-corrected multiple comparisons). Theweight loss profile in glycan-treated animals was not significantlydifferent than that of the vancomycin-treated group.

B. Effect of Treatments on Carriage of C. difficile Spores.

On days 0-6 the presence of C. difficile spores in the feces of mice wasassessed. Vancomycin-treated animals had no detectable spores in theirfeces on any day. Mice that were treated with glycans or water hadbetween 2 and 6 log₁₀ CFU/gram feces. Despite carriage of C. difficilespores on day 6, the glycan-treated animals remained healthy through day10 with clinical scores on days 7 and 10 of 0. All vancomycin-treatedanimals exhibited clinical signs on day 10, with 17% deaths.

C. Glycan-Associated Shifts in Microbial Gut Constituents

To determine how the gut microbiota composition responds to treatmentwith glycan therapeutics, 16S rRNA sequencing of fecal pellets on the V4variable region using standard protocols from the Earth MicrobiomeProject was performed (www.earthmicrobiome.org/emp-standard-protocols/).

On day 6 after the termination of glycan treatment, there was asignificant difference between the gut microbiota phylogeneticcomposition of mice treated with glycans (L or S) and either FOS orvehicle and vancomycin controls (Table 11).

TABLE 11 Comparison of ending composition between glycan treatments withvehicle, vancomycin, or FOS controls. Vehicle Vancomycin FOS Glu100 —— * Glu33gal33fuc33 ** ** * Glu50gal50 *** *** *** Ara100 ** ** * *adj.P < 0.05, **adj. P < 0.01, ***adj. P < 0.001; pairwise adonis,Bonferonni-corrected multiple comparisons

Different bacterial genera were increased in relative abundance withvarious glycan treatments, vancomycin treatment, or water control. FIG.12 shows changes in relative abundance of specific bacterial genera fromdirectly before (Day −1) to directly after (Day 6) treatment withglycans or vancomycin. The genus Bacteroides was increased in ara100,FOS, and glu50gal50 treatments. The genus Parabacteroides was increasedin glu33gal33fuc33 treatments. The genus Bifidobacterium was increasedin FOS, glu100, and glu50gal50 treatments. Bacteroides, Parabacteroides,and Bifidobacterium have been shown to positively correlate with a C.difficile resistant microbiome composition (Seekatz and Young, 2014, TheJournal of Clinical Investigation). Notably, the genera Enterococcus wasdecreased in all treatments and Lactobacillus was decreased in FOS,glu100, and glu50gal50 treatments. Enterococcus and Lactobacillus haveboth been previously observed to be positively correlated with a C.difficile susceptible microbiome composition (Seekatz and Young, 2014,The Journal of Clinical Investigation).

Some secondary bile acids have been shown to impair C. difficile growthin vitro. The capacity for the gut microbiome to convert primary bileacids to secondary bile acids has been hypothesized to directlyantagonize C. difficile infection. Three glycans (glu33gal33fuc33,ara100, and glu50gal50) all increased the predicted functional potentialof the gut microbiome to convert primary bile acids to secondary bileacids (FIG. 13). This was not observed in vancomycin controls. Inaddition, one glycan (glu100) and commercially available FOS bothdecreased the predicted functional potential of the gut microbiome onaverage. Functional predictions were made from 16S rRNA sequencing usingPICRUST (picrust.github.io/picrust/).

Secondary bile acid production is linked to reduced germination andgrowth of C. difficile, and members of the Lachnospiraceae andRuminococcaceae families have been associated with secondary bile acidproduction and resistance to C. difficile germination and growth(Theriot C M, Bowman A A and Young V B. 2016. Antibiotic-inducedalterations of the gut microbiota alter secondary bile acid productionand allow for Clostridium difficile spore germination and outgrowth inthe large intestine. mSphere 1(1):e00045-15.) Glycan therapeutics whenadministered in an effective amount may modulate bacterial species thatproduce secondary bile acids and thereby promote resistance to C.difficile germination, outgrowth and colonization.

In the in vitro assay of Example 8, growth of ROB.74, a member of theRuminocacceae family, was supported by 13 out of 15 glycans, and growthof CSC.32 and CNE.31, members of the Lachnospiraceae family, weresupported by 6 and 7 out of 15 glycans, respectively, as shown in Table8. Two glycan treatments, glu50gal50 and ara100, significantly increasedthe relative abundance of the secondary bile acid biosynthesis pathwayin the mouse model of C. difficile infection (FIG. 13).

The alpha diversity (as measured by Shannon index) of the gut microbiotaall increased directly before (Day −1) to directly after (Day 6)treatment with glycan (FIG. 14).

These results, obtained in a widely used animal model for Clostridiumdifficile infection suggest that glycan therapeutics reduced C.difficile-induced weight loss and improved survival. Selected glycansappear to promote the growth of bacterial genera that contribute to C.difficile resistance (Bacteroides, Parabacteroides, and Bifidobacterium)and conditions that are less favorable for C. difficile growth (e.g.increased presence of secondary bile acids).

Example 13 Effect of Glycans in a Mouse Model of Colitis

This experiment was conducted to analyze the effects of glycantherapeutics in a mouse model of colitis (Okayasu et al, 1990, A NovelMethod in the Induction of Reliable Experimental Acute and ChronicUlcerative Colitis in Mice. Gastroenterology 98:694-702). In this model,normal mice were exposed to dextran sulfate sodium (DSS) in theirdrinking water which induced an onset of disease symptoms similar withrespect to their development and manifestation in human clinicaldisease. Human ulcerative colitis is a progressive, inflammatorydisorder of the intestinal tract with a prevalence of about 150-300 per100,000 people. Ulcerative colitis is a result of an aberrant hostimmune response to commensal flora, is limited to the colon and involvesdiffuse mucosal inflammation. Symptoms in humans include colonicinflammation/ulceration, weight loss and diarrhea which may containblood.

Mice (male C57BL/6, 6-8 weeks old, 22-24 grams each; Charles RiverLaboratories, Wilmington Mass.) were housed in groups of 8 per cage,with 1 cage per treatment group. The vehicle control group notadministered 2.5% DSS was comprised of 6 mice. Mice were administeredglycan therapeutics at 1% concentration in their drinking water from day−7 (7 days prior to DSS administration) to day 14. On days 0 to 5 micewere administered DSS (MP Bio, Santa Ana Calif.; 36,000-50,000 Da) at2.5% in their drinking water to induce colitis phenotypes.

Disease-associated phenotypes were recorded for a duration of twelvedays starting on day 0 through day 11. The phenotypes that were scoredwere: weight, incidence of diarrhea and blood in stool. In addition,endoscopic analysis of colon inflammation was performed on day 14. Thefollowing scoring system was used: Naïve, no edema or mucosal sloughing,clear vascularity=0; edema, mucosal sloughing, decreased vascularity=1;edema, mucosal sloughing, decreased vascularity, friability=2; activebleeding, erosion=3; active ulceration, erosion=4. Histopathologicanalysis was undertaken on terminal colon samples of all of the mice.Colon sections were fixed in formalin and stained with hematoxylin andeosin (H&E). A semi-quantitative analysis of colonic lesions includingmucosal erosion, loss of colonic glands, and mucosal to transmuralinflammation with regenerating (hyperplastic) mucosal and glandularepithelium was used. All of the phenotypes mimic those of human colitis.In this model peak weight loss generally occurs at day 10.

All animals were administered 2.5% DSS in the drinking water for days0-5. The following glycan treatments were delivered in the animals'drinking water at a concentration of 1%: FOS (commercial, control);glu100; man52glu29gal19; acacia fiber (commercial, control) from day −7to day 14. The control group received plain drinking water in place ofglycan containing water.

Effects of test treatments was assessed by comparing thedisease-associated phenotypes in glycan-treated and untreated (plainwater) groups. FOS (Nutraflora FOS; NOW Foods, Bloomingdale Ill.) is acommercially-available non-digestible fructooligosaccharide. Acaciafiber (Organic Powder, NOW Foods, Bloomingdale, Ill.) is acommercially-available fiber supplement.

Treatment with glu100 and man52glu29gal19 reduced the incidence ofdiarrhea, and weight loss from day 0 to 11 (FIG. 15), as well assignificantly reduced colonic inflammation (day 14; FIG. 16) as assessedby endoscopy in mice as compared with either plain water (vehiclecontrol) or acacia fiber-treated animals. Histopathological results ofcolon samples from the mice mirrored those from endoscopic analysis withregard to glycan efficacy. Cumulative days of diarrhea incidence in thecontrol and treatment groups were as follows: water control group: 10days; acacia fiber: 14 days; FOS: 4 days; glu100: 2 days;man52glu29gal19: 3 days. Taken together these data demonstrate thatglycan treatments have significant protective effects in a mouse modelof colitis.

These results, obtained in a widely used animal model of colitis,suggest that glycan therapeutics reduce DSS-induced weight loss,diarrhea and colonic inflammation and damage as assessed by endoscopyand histopathology.

Example 14 Effect of Glycans in a Mouse Model of Diet-Induced Obesity

This experiment was conducted to analyze the effects of glycantherapeutics in a mouse model of high fat diet-induced obesity (Wang andLiao, A Mouse Model of Diet-Induced Obesity and Insulin Resistance.Methods Mol Biol. 2012; 821: 421-433). In this model, normal mice werefed a diet containing 60% fat content over 6 weeks. These mice exhibiteda significant increase in weight compared with mice fed a low (10%) fatdiet. In humans, obesity is the accumulation of excess body fat to theextent that it may have a negative effects on health, including thedevelopment of type 2 diabetes and cardiovascular disease. The majorcauses of obesity are excessive food energy intake, lack of physicalactivity, and genetic predisposition. In the US, about 38% of adults (78million) and 17% of children and adolescents (13 million) are obese. Themouse model of diet-induced obesity recapitulates human diseaseendpoints including weight gain, decreased lean body mass, changes inorgan physiology and changes in markers of diabetes.

Mice (male C57BL/6, 8-10 weeks old, 16-18 grams each; Taconic Labs,Germantown, N.Y.) were housed in groups of 1-2 per cage, with 8 animalsper treatment group. Mice were fed a high (60%) fat diet (60% of totalkcal contributed by fat) (D12492; Research Diets, New Brunswick, N.J.)or matched diet containing 10% fat (10% of total kcal contributed byfat) (D12450; Research Diets). After one week on this diet regimen, miceon high fat diet were administered glycans (self-administered indrinking water; day 0 to day 44): glu100 at 0.3% weight/volume (w/v) orman52glu29gal19 at 1% w/v in drinking water, commercial FOS (NutrafloraFOS; NOW Foods, Bloomingdale Ill.) at 6%, 1% or 0.3% w/v in drinkingwater, or plain water (control). The group of mice consuming the low fatdiet was given plain water (control). Diets were kept the same for eachgroup throughout the study.

Weight Gain

Weight was monitored every other day from day 0 through day 44. Mice ona high fat diet gained weight at a significantly faster pace than micefed a low fat diet. Treatment with glu100 (0.3% in drinking water) andman52glu29gal19 (1% in drinking water) significantly decreased the slopeof the weight gain curve from day 0 to 41 in high fat-fed mice ascompared with water (vehicle)-treated mice (FIG. 17A and FIG. 17B). FOSat any dose had no significant effect on the percent (%) weight changeslope as compared with water-control animals.

Glucose Tolerance

On day 42, mice were subjected to an oral glucose tolerance test byfasting them for 12 hours and then administering an orally-gavaged doseof glucose (2 grams/kg; Ayala et. al. Standard operating procedures fordescribing and performing metabolic tests of glucose homeostasis in miceDisease Models & Mechanisms 3, 525-534 (2010)). Blood glucose levelswere monitored using a handheld glucometer (One Touch Ultra®; LifeScanInc, Wayne Pa.) at 120 minutes post glucose dose. Mice on a high fatdiet had a lower ability to clear glucose from their systemiccirculation than mice fed a low fat diet (FIG. 18). High fat diet-fedmice treated with glu100 (0.3%) and man52glu29gal19 (1%) had lower bloodglucose levels as compared to high fat diet-fed, water-control mice.These levels were more similar to the low fat diet-fed, water-controlmice.

Fat Pad Development

On day 44, the epididymal fat pads of the mice were removed and weighed.The weight of the fat pads as a percent of total body weight was used asa surrogate endpoint for lean body mass, with an increased fat padweight corresponding to lower lean body mass. Mice treated withman52glu29gal19 (1%) and fed the high fat diet had a lower fat pad/bodyweight % than water-control mice (FIG. 19) or FOS-treated mice fed thehigh fat diet.

The results for these three endpoints in the obesity model suggest thata longer term model could lead to significant changes in otherparameters that are assessed in the model (Wang and Liao, A Mouse Modelof Diet-Induced Obesity and Insulin Resistance. Methods Mol Biol. 2012;821: 421-433). For example, high fat diet-fed mice will exhibit multipleorgan changes when compared with low-fat diet fed mice. In the 12^(th)week of the experiment and beyond, mice are sacrificed and certaintissues harvested/processed for analysis. The colon and cecum arecollected, weighed and measured; obese mice will have a shorter colonand cecum and colon of decreased weight. The cecal contents are snapfrozen for storage for subsequent analysis of 16S RNA and metabolites.Livers are collected, weighed and stored in formalin for histopathology.Mice fed a high fat diet tend to have higher liver weights than thosefed a low fat diet. Blood samples are taken and processed to plasma formeasurement of inflammatory markers (TNF-α, IFN-γ, IL-10, IL-13, IL-1β,IL-4, IL-5, IL-6 and KC/GRO), clinical chemistry (cholesterol,triglycerides), and lipopolysaccharide (LPS) levels. Mice fed a high fatdiet have increased levels of markers of inflammation, and highercholesterol, triglyceride and circulating LPS levels. All of theseendpoints in the model test similar manifestations of obesity as areobserved in humans. Selected glycans reduce the incidence or magnitudeof shorter colon and cecum and decreased colon weight. Selected glycansreduce the incidence or magnitude of liver weight gain. Selected glycansreduce the incidence or magnitude of presence of inflammatory markers,reduce cholesterol and/or systemic LPS levels.

These results, obtained in a widely used animal model for diet-inducedobesity suggest that glycan therapeutics may prevent high fatdiet-induced weight gain, improve the ability to clear blood glucose andincrease lean/fat body composition.

Example 15 Effect of Glycans on Gene Expression in a Mouse Model

The trial is conducted with two groups of mice. The control group ofmice are fed with standard chow, and the different treatment groups ofmice are fed with standard chow supplemented with glycans. After 1-30days, blood samples are drawn from the mice, the mice are sacrificed,and tissues from the intestine, liver, skin, and other sites of interestare collected and stored at −80° C. RNA is isolated from the tissues andconverted to cDNA. The GeneChip Mouse Genome 430 2.0 Array (Affymetrix)is used to analyze the differential expression between the untreated andglycan-treated animals of approximately 14,000 murine genes. Theexperimental protocol and raw data analysis are performed according tothe manufacturer's instructions and standard protocols. The biologicalfunction of the differentially expressed genes and their involvement invarious processes are obtained from the following databases: RefGene(Reference for genes, proteins and antibodies: refgene.com/), CTD(Comparative Toxicogenomics Database: ctd.mdibl.org/), MGI (MouseGenomics Informatics: www.informatics.jax.org/), KEGG (KyotoEncyclopedia of Genes and Genomes: www.genome.jp/kegg/genes.html). Thisprocedure is used to identify the differential expression of genesencoding inflammatory cytokines, immunomodulatory cytokines,antimicrobial peptides, and other relevant effector molecules.

TABLE 1 Genus level Microbial Constituents of the GI tract. Phylum ClassGenus Actinobacteria Actinobacteria Actinomyces, Adlercreutzia,Atopobium, Bifidobacterium, Collinsella, Corynebacterium, Eggerthella,Mobiluncus, Propionibacterium, Rothia, Slackia Bacteroidetes BacteroidiaAlistipes, Bacteroides, Dysgonomonas, Odoribacter, Parabacteroides,Porphyromonas, Prevotella, Tannerella Flavobacteria CapnocytophagaFirmicutes Bacilli Bacillus, Enterococcus, Gemella, Granulicatella,Lactobacillus, Lactococcus, Staphylococcus, Streptococcus, Turicibacter,Weissella Clostridia Acidaminococcus, Anaerococcus, Anaerofilum,Anaerofustis, Anaerostipes, Anaerotruncus, Anaerovorax, Bacteroides,Bacteroides, Blautia, Clostridium, Coprococcus, Dehalobacterium,Dialister, Dorea, Eubacterium, Faecalibacterium, Finegoldia,Lachnobacterium, Lachnospira, Megamonas, Megasphaera, Mitsuokella,Moryella, Oribacterium, Oscillospira, Peptococcus, Peptoniphilus,Peptostreptococcus, Phascolarctobacterium, Pseudobutyrivibrio,Roseburia, Ruminococcus, Ruminococcus, Selenomonas, Subdoligranulum,Veillonella Fusobacteria Fusobacteria Fusobacterium, LeptotrichiaBetaproteobacteria Comamonas, Herbaspirillum, Lautropia, Neisseria,Oxalobacter, Sutterella Deltaproteobacteria Bilophila, Desulfovibrio,LE30 Epsilonproteobacteria Campylobacter, HelicobacterGammaproteobacteria Actinobacillus, Aggregatibacter, Citrobacter,Escherichia, Haemophilus, Klebsiella, Moraxella, Pseudomonas, RaoultellaSpirochaetes Spirochaetes Treponema Synergistetes SynergistetiaCloacibacillus, Synergistes Tenericutes Erysipelotrichi Bulleidia,Catenibacterium, Clostridium, Coprobacillus, Holdemania, RFN20Mollicutes Asteroleplasma, Mycoplasma Verrucomicrobia VerrucomicrobiaeAkkermansia Euryarchaeota Methanobacteria Methanobrevibacter

TABLE 2 Microbial Metabolites 2-hydroxyisobutyrate,3-hydroxyisovalerate, 3-methyl-crotonylglycine, 3-methylcrotonylglycine, allantoin, betaine, formate, mannitol, p-cresolglucuronide, phenylacetylglycine, sarcosine, taurine, acetic acid,acetylaldehyde, ascorbic acid, butanedione, butyric acid, deoxycholicacid, ethylphenyl sulfate, formic acid/formate, indole, isobutyric acid,isovaleric acid, propionic acid, serotonin, succinic acid/succinate,TMAO, tryptophan, valeric acid, ursodeoxycholic acid, lactate, lacticacid, hydrogen peroxide

TABLE 3 Genus level microbial constituents predominant in the largeintestine (compared to small intestine) in healthy humans. Phylum ClassGenus Bacteroidetes Bacteroidia Bacteroides, Butyricimonas, Odoribacter,Parabacteroides, Prevotella Firmicutes Clostridia Anaerotruncus,Phascolarctobacterium, Ruminococcus, Proteobacteria DeltaproteobacteriaBilophila Verrucomicrobia Verrucomicrobiae Akkermansia

TABLE 4 Genus level microbial constituents predominant in the smallintestine (compared to large intestine) in healthy humans. Phylum ClassGenus Actinobacteria Actinobacteria Cryocola, Mycobacterium FirmicutesBacilli Enterococcus, Lactococcus, Streptococcus, TuricibacterClostridia Blautia, Coprococcus, Holdemania, PseudoramibacterEubacterium Proteobacteria Alphaproteobacteria Agrobacterium,Sphingomonas Betaproteobacteria Achromobacter, Burkholderia, Ralstonia

TABLE 5 Polyphenols Polyphenol Sub- Class Compound Name AnthocyaninsMalvidin 3-O-(6″-p-coumaroyl-glucoside), Cyanidin, total, Delphinidin3-O- (6″-acetyl-galactoside), Cyanidin 3-O-(6″-acetyl-galactoside),Malvidin, Cyanidin 3-O-galactoside, Cyanidin 3-O-glucoside, Cyanidin3-O-rutinoside, Cyanidin 3-O-sophoroside, Pelargonidin 3-O-glucoside,Cyanidin 3-O-(6″- malonyl-glucoside), Peonidin, Peonidin 3-O-glucoside,Peonidin 3-O- rutinoside, Pelargonidin 3-O-rutinoside, Pelargonidin,Cyanidin, Malvidin 3,5-O-diglucoside, Cyanidin 3-O-glucosyl-rutinoside,Pelargonidin 3-O- sophoroside, Pelargonidin 3-O-glucosyl-rutinoside,Cyanidin 3-O-(6″- succinyl-glucoside), Pelargonidin3-O-(6″-succinyl-glucoside), Delphinidin, Delphinidin 3-O-galactoside,Delphinidin 3-O-glucoside, Delphinidin 3-O- arabinoside, Petunidin,Petunidin 3-O-galactoside, Cyanidin 3-O-arabinoside, Petunidin3-O-glucoside, Peonidin 3-O-galactoside, Petunidin 3-O- arabinoside,Malvidin 3-O-glucoside, Malvidin 3-O-arabinoside, Cyanidin 3-O-(6″-acetyl-arabinoside), Delphinidin 3-O-(6″-acetyl-glucoside),Petunidin 3-O-(6″-acetyl-galactoside), Peonidin3-O-(6″-acetyl-galactoside), Cyanidin 3-O-(6″-acetyl-glucoside),Malvidin 3-O-(6″-acetyl-galactoside), Petunidin 3-O-(6″-acetyl-glucoside), Polymeric anthocyanins, total, Malvidin3-O-(6″- acetyl-glucoside), Peonidin 3-O-(6″-acetyl-glucoside),Pelargonidin 3-O- arabinoside, Delphinidin 3-O-rutinoside, Cyanidin3-O-sambubioside, Pelargonidin 3-O-(6″-malonyl-glucoside), Peonidin3-O-(6″-p-coumaroyl- glucoside), Cyanidin 3-O-xyloside, Malvidin3-O-galactoside, Peonidin 3-O- arabinoside, Petunidin 3-O-rutinoside,Delphinidin 3-O-xyloside, Petunidin 3-O-(6″-p-coumaroyl-glucoside),Pelargonidin 3-O-galactoside, Pelargonidin 3-O-sambubioside, Delphinidin3-O-sambubioside, Cyanidin 3-O-xylosyl- rutinoside, Vitisin A,Delphinidin 3-O-(6″-p-coumaroyl-glucoside), Pigment A, p-Coumaroylvitisin A, Acetyl vitisin A, Cyanidin 3-O-(6″-p-coumaroyl- glucoside),Cyanidin 3-O-sambubioside 5-O-glucoside, Cyanidin 3-O-(6″-caffeoyl-glucoside), Cyanidin 3,5-O-diglucoside, Pinotin A, Delphinidin3,5- O-diglucoside, Pelargonidin 3,5-O-diglucoside, Malvidin3-O-(6″-caffeoyl- glucoside), Cyanidin 3-O-(6″-dioxalyl-glucoside),Cyanidin 3-O- laminaribioside, Cyanidin 3-O-(3″-malonyl-glucoside),Peonidin 3-O-(6″- malonyl-glucoside), Cyanidin3-O-(6″-malonyl-laminaribioside), Cyanidin 3-O-dimalonyl-laminaribioside, Cyanidin 3-O-(6″-malonyl-arabinoside),Delphinidin 3-O-glucosyl-glucoside, Cyanidin3-O-(6″-malonyl-3″-glucosyl- glucoside), Cyanidin3-O-(2″-xylosyl-6″-glucosyl-galactoside), Cyanidin 3-O-(2″-xylosyl-6″-(6″′-caffeoyl-glucosyl)-galactoside), Cyanidin 3-O-(2″-xylosyl-galactoside), Cyanidin 3-O-(2″-xylosyl-6″-(6″′-p-hydroxybenzoyl-glucosyl)-galactoside), Cyanidin3-O-(2″-xylosyl-6″-(6″′-sinapoyl-glucosyl)- galactoside), Cyanidin3-O-(2″-xylosyl-6″-(6″′-feruloyl-glucosyl)- galactoside), Cyanidin3-O-(2″-xylosyl-6″-(6″′-p-coumaroyl-glucosyl)- galactoside), Delphinidin3-O-(6″-malonyl-glucoside), Malvidin 3-O- rutinoside, Luteolinidin3-O-glucoside, Delphinidin 3-O-feruloyl-glucoside, Petunidin3,5-O-diglucoside, Petunidin 3-O-rhamnoside, Luteolinidin, Vitisin Aaglycone, Pigment A aglycone, Pinotin A aglycone, 4-O-Methylcyanidin3-O-galactoside, Malvidin 3-O-(6″-O-acetyl)-glucoside, Cyanidin 3-O-diglucoside-5-O-glucoside, Peonidin 3-O-diglucoside-5-O-glucoside,Peonidin 3,5-O-diglucoside, Peonidin 3-O-(2-O-(6-O-(E)-caffeoyl-D-glucosyl)-D-glucoside)-5-O-D-glucoside, Peonidin 3-O-sophoroside,Peonidin 3-O-sambubioside, Peonidin 3-O-sambubioside-5-O-glucoside,Peonidin 3-O-xyloside, 4′-O-Methylcyanidin 3-O-D-glucoside, Cyanidin 3-O-glucuronide, Cyanidin 3-O-(3″,6″-O-dimalonyl-glucoside), Cyanidin 3-sulfate, 4-O-Methyldelphinidin 3-O-L-arabinoside, 4-O-Methyldelphinidin3- O-D-glucoside, Isopeonidin 3-O-arabinoside, Isopeonidin3-O-galactoside, Isopeonidin 3-O-glucoside, Isopeonidin 3-O-rutinoside,Isopeonidin 3-O- sambubioside, Isopeonidin 3-O-xyloside,4-O-Methylpetunidin 3-O-D- galactoside, 4-O-Methylpetunidin3-O-D-glucoside, Cyanidin 3-O-(2-O-(6- O-(E)-caffeoyl-Dglucoside)-D-glucoside)-5-O-D-glucoside, 4′-O- Methyldelphinidin3-O-rutinoside, Pelargonidin 3-O-(6″-acetyl-glucoside) ChalconesChalconaringenin, total, Butein, Xanthohumol, Chalconaringenin,Chalconaringenin 2′-O-glucuronide, Chalconaringenin 4′-O-glucuronide,Chalconaringenin 7-O-glucuronide _Dihydro- Phloretin, Phloridzin,Phloretin xylosyl-galactoside, Phloretin 2′-O-xylosyl- chalconesglucoside, 3-Hydroxyphloretin 2′-O-xylosyl-glucoside, 3-Hydroxyphloretin2′-O-glucoside, Phloridzin, total, 3-Hydroxyphloretin, Phloretin 2′-O-glucuronide, 3-Methoxyphloretin 3*-O-glucoside, 3-Hydroxy-4-O-methylphloretin 3*-O-glucoside, 3-Hydroxyphloretin 3*-O-glucosideDihydro- Dihydroquercetin 3-O-rhamnoside, Dihydroquercetin, Engeletin,flavonols Dihydromyricetin 3-O-rhamnoside, Dihydroquercetin3-O-glucoside, Dihydromyricetin, Dihydrokaempferol Flavanols(+)-Catechin, (−)-Epicatechin, (+)-Gallocatechin, (−)-Epigallocatechin,(−)- Epicatechin 3-O-gallate, (−)-Epigallocatechin 3-O-gallate,Catechins, total, Theaflavins, total, Thearubigins, total, Theaflavin,Theaflavin 3-O-gallate, Theaflavin 3*-O-gallate, Theaflavin3,3*-O-digallate, (+)-Gallocatechin 3-O- gallate, (−)-Catechin,(+)-Catechin 3-O-gallate, Theaflavic acid, Epitheaflavic acid,Epitheaflavic acid 3*-O-gallate, Isoneotheaflavin 3-O-gallate, (−)-Gallocatechin 3-O-gallate, (−)-Gallocatechin, (−)-Catechin 3-O-gallate,(+)- Epicatechin, (−)-Epicatechin 8-C-galactoside, Isoneotheaflavin,Procyanidin dimer Bl, Procyanidin dimer B2, Procyanidin dimer B3,Procyanidin dimer B4, Procyanidin dimer B5, Procyanidin dimer B7,Prodelphinidin dimer B3, Procyanidin trimer C1, Procyanidin tetramer T4,02 mers, Procyanidins, total, Procyanidin trimer EEC, 01 mers, Polymers(>10 mers), 03 mers, 04-06 mers, 07-10 mers, Procyanidin dimer B6,Procyanidin trimer T2, Procyanidin trimer C2, Procyanidin dimer B23-O-gallate, Procyanidin dimer B2 3′-O- gallate, Procyanidin dimer B13-O-gallate, Prodelphinidin trimer GC-GC-C, Procyanidin trimer T3, 04mers, Procyanidin dimer A2, 05 mers, 06 mers, 07 mers, 08 mers, 09 mers,10 mers, 02-03 mers, (+)-Epicatechin-(2a-7)(4a-8)- catechin3-O-arabinoside, Cinnamtannin B1 3-O-galactoside, (+)-Epicatechin-(2a-7)(4a-8)-epicatechin 3-O-arabinoside, Cinnamtannin B1 3-O-arabinoside, Procyanidin dimer A1, Cinnamtannin B1, Proanthocyanidins,total, Prodelphinidin trimer GC-C-C, Prodelphinidin trimer C-GC-C, (+)-Epicatechin-(2a-7)(4a-8)-catechin,(+)-Epicatechin-(2a-7)(4a-8)-epicatechin,(−)-Epicatechin-(2a-7)(4a-8)-epicatechin 3-O-galactoside, CinnamtanninA2, Bis-8,8′-Catechinylmethane, Cinnamtannin A3, (+)-Catechin3-O-glucose, 3′- O-Methylepicatechin, 4′-O-Methyl-(−)-epicatechin3*-O-glucuronide, Epicatechin 3*-O-glucuronide, Epigallocatechin3-O-gallate-4″-O- glucuronide, 3′-O-Methylcatechin,3′-O-Methyl-(−)-epicatechin 3-O-gallate,4′,4″-O-Dimethylepigallocatechin 3-O-gallate,4′-O-Methylepigallocatechin, 4″-O-Methylepigallocatechin 3-O-gallate,4′-O-Methylepicatechin, Epigallocatechin3-O-gallate-7-O-glucoside-4″-O-glucuronide, Theasinensin A,3-O-Methylepigallocatechin, 3′,4″-Dimethyl-(−)-epicatechin 3-O-gallate,(−)-Epigallocatechin 3-O-glucuronide, 3′-O-Methyl-(−)-epigallocatechin3-O- gallate, 3″-O-Methyl-(−)-epigallocatechin 3-O-gallate,3′,3″-O-Dimethyl-(−)- epigallocatechin 3-O-gallate,3′-O-Methyl-(−)-epicatechin 7-O-glucuronide, Epicatechin7-O-glucuronide, (−)-Epigallocatechin 3*-O-glucuronide, (−)-Epigallocatechin 7-O-glucuronide, 4′-O-Methyl-(−)-epigallocatechin 3*-O-glucuronide, 4′-O-Methyl-(−)-epigallocatechin 7-O-glucuronide,4′-O-Methyl- (−)-epigallocatechin 3′-sulfate Flavanones Naringenin,Eriodictyol, Hesperetin, Hesperetin, total, Naringenin, total,Eriocitrin, Hesperidin, Naringin, Narirutin, Neoeriocitrin,Neohesperidin, Isosakuranetin 7-O-rutinoside, Poncirin, Didymin,Narirutin 4′-O-glucoside, Naringin 4′-O-glucoside, Naringin 6′-malonate,Isosakuranetin, Naringenin 7- O-glucoside, Pinocembrin,8-Prenylnaringenin, 6-Prenylnaringenin, 6- Geranylnaringenin,Isoxanthohumol, Eriodictyol 7-O-glucoside, Sakuranetin, Hesperetin3*-O-glucuronide, Hesperetin 7-O-glucuronide, Hesperetin 3′- sulfate,Hesperetin 7-sulfate, Homoeriodictyol, Naringenin 4′-O-glucuronide,Naringenin 5-O-glucuronide, Naringenin 7-O-glucuronide, Hesperetin3′,7-O- diglucuronide, Hesperetin 5,7-O-diglucuronide, Pinobanksin, 5-O-Methylpinobanksin Flavones Apigenin, Luteolin, Apigenin, total,Luteolin, total, Diosmin, Isorhoifolin, Neodiosmin, Rhoifolin,Sinensetin, Nobiletin, Tangeretin, Luteolin 7-O- diglucuronide, Chrysin,Diosmetin, Acacetin, Luteolin 7-O-rutinoside, Tetramethylscutellarein,Luteolin 7-O-glucoside, Apigenin 7-O-glucoside, Apigenin6,8-di-C-glucoside, Sinensetin, total, Apigenin 6,8-C-arabinoside-C-glucoside, Apigenin 6,8-C-galactoside-C-arabinoside, Luteolin 7-O-glucuronide, Apigenin 7-O-glucuronide, Luteolin 7-O-malonyl-glucoside,Luteolin 6-C-glucoside, Luteolin 8-C-glucoside, Luteolin 6-C-glucoside8-C- arabinoside, Luteolin 7-O-(2-apiosyl-glucoside), Luteolin7-O-(2-apiosyl-4- glucosyl-6-malonyl)-glucoside, Apigenin 6-C-glucoside8-C-arabinoside, Luteolin 7-O-(2-apiosyl-6-malonyl)-glucoside, Apigenin7-O-apiosyl- glucoside, Apigenin 8-C-glucoside,7,3′,4′-Trihydroxyflavone, 7,4′- Dihydroxyflavone, Geraldone, Baicalein,Apigenin 6-C-glucoside, Hispidulin, Cirsimaritin, Luteolin4′-O-glucoside, 5,6-Dihydroxy-7,8,3′,4′- tetramethoxyflavone, Pebrellin,Gardenin B, Nepetin, Jaceosidin, Cirsilineol, Eupatorin,6-Hydroxyluteolin, 6-Hydroxyluteolin 7-O-rhamnoside, Scutellarein,Apigenin 7-O-(6″-malonyl-apiosyl-glucoside), Chrysoeriol, Chrysoeriol7-O-apiosyl-glucoside, Chrysoeriol 7-O-(6″-malonyl-apiosyl- glucoside),Chrysoeriol 7-O-glucoside, Chrysoeriol 7-O-(6″-malonyl- glucoside),Apigenin 7-O-diglucuronide, Rhoifolin 4′-O-glucoside, 3′-O-Demethylnobiletin, 4′-O-Demethylnobiletin, 6-O-Demethyleupatilin, 6-O-Methylscutellarin, Apigenin 4′-O-glucuronide, Apigenin 5-O-glucuronide,Eupatilin, Isoscutellarein, Scutellarein 4′-O-glucuronide, Scutellarein5-O- glucuronide, Scutellarein 6,7-O-diglucuronide, Scutellarein6-O-glucuronide, Scutellarein 7-sulfate, Scutellarein 7-O-glucuronide,Tricin, 6-O- Methylscutellarein Flavonols Kaempferol, Quercetin,Quercetin 3-O-galactoside, Quercetin 3-O-glucoside, Quercetin3-O-xyloside, Quercetin 3-O-rhamnoside, Quercetin 3-O- rutinoside,Quercetin 3-O-sophoroside, Quercetin 3-O-arabinoside, Quercetin3-O-xylosyl-glucuronide, Quercetin, total, Kaempferol, total, Myricetin,total, Isorhamnetin 3-O-glucoside 7-O-rhamnoside, Isorhamnetin3-O-rutinoside, Kaempferol 3-O-glucuronide, Isorhamnetin 7-O-rhamnoside,Quercetin 3,4′- O-diglucoside, Myricetin 3-O-rutinoside, Myricetin,Morin, Kaempferide, Myricetin 3-O-galactoside, Myricetin 3-O-glucoside,Quercetin 3-O- glucosyl-xyloside, Quercetin 3-O-acetyl-rhamnoside,Kaempferol 3-O- galactoside, Galangin, Isorhamnetin, Kaempferol3-O-glucoside, Kaempferol 3-O-rutinoside, Kaempferol3-O-glucosyl-rhamnosyl-galactoside, Kaempferol3-O-glucosyl-rhamnosyl-glucoside, Quercetin 3-O-glucosyl-rhamnosyl-galactoside, Quercetin 3-O-glucosyl-rhamnosyl-glucoside,Rhamnetin, Isorhamnetin 3-O-glucoside, Myricetin 3-O-rhamnoside,Quercetin 3-O-rhamnosyl-galactoside, Kaempferol 3-O-arabinoside,Quercetin 3-O-glucuronide, Isorhamnetin 3-O-glucuronide, Myricetin 3-O-arabinoside, Quercetin 3,7,4′-O-triglucoside, Quercetin7,4′-O-diglucoside, Quercetin 4′-O-glucoside, Isorhamnetin4′-O-glucoside, 3,7- Dimethylquercetin, Kaempferol 3-O-sophoroside,Kaempferol 3,7-O- diglucoside, Quercetin 3-O-diglucoside, Kaempferol3-O-sophoroside 7-O- glucoside, Kaempferol 3-O-sophorotrioside7-O-sophoroside, Kaempferol 3- O-sinapoyl-caffeoyl-sophoroside7-O-glucoside, Kaempferol 3-O-feruloyl- caffeoyl-sophoroside7-O-glucoside, Kaempferol 3-O-feruloyl- sophorotrioside, Kaempferol3-O-sinapoyl-sophoroside 7-O-glucoside, Kaempferol3-O-caffeoyl-sophoroside 7-O-glucoside, Kaempferol 3-O-feruloyl-sophoroside 7-O-glucoside, Quercetin3-O-(6″-malonyl-glucoside), Kaempferol 3-O-(6″-malonyl-glucoside),Kaempferol 3-O-rhamnoside, Quercetin 3-O-(6″-malonyl-glucoside)7-O-glucoside, Patuletin, Quercetagetin, Spinacetin, Patuletin3-O-glucosyl-(1->6)-[apiosyl(1->2)]- glucoside, Spinacetin3-O-glucosyl-(1->6)-[apiosyl(1->2)\-glucoside, Patuletin3-O-(2″-feruloylglucosyl)(1->6)-[apiosyl(1->2)\-glucoside, Spinacetin3-O-(2″-p-coumaroylglucosyl)(1->6)-[apiosyl(1->2)\-glucoside, Spinacetin3-O-(2″-feruloylglucosyl)(1->6)-[apiosyl(1->2)\-glucoside, Spinacetin3-O-glucosyl-(1->6)-glucoside, Jaceidin 4′-O-glucuronide, 5,3′,4′-Trihydroxy-3-methoxy-6:7-methylenedioxyflavone 4′-O-glucuronide, 5,4′-Dihydroxy-3,3′-dimethoxy-6:7-methylenedioxyflavone 4′-O-glucuronide,Spinatoside, Spinatoside 4′-O-glucuronide, Kaempferol 3-O-xylosyl-glucoside, Kaempferol 3-O-acetyl-glucoside, Quercetin 3-O-xylosyl-rutinoside, Kaempferol 3-O-xylosyl-rutinoside, Quercetin 3-O-glucosyl-glucoside, Quercetin 7-O-glucoside, Quercetin 3-O-(6″-acetyl-glucoside),Kaempferol 3-O-robinoside 7-O-rhamnoside, Kaempferol 7-O-glucoside,Kaempferol 3-O-galactoside 7-O-rhamnoside, Kaempferol 3-O-(6″-acetyl-galactoside) 7-O-rhamnoside, Quercetin 3-O-galactoside 7-O-rhamnoside,Quercetin 3-O-(6″-acetyl-galactoside) 7-O-rhamnoside, Kaempferol3-O-(2″- rhamnosyl-galactoside) 7-O-rhamnoside, Kaempferol3-O-(2″-rhamnosyl-6″- acetyl-galactoside) 7-O-rhamnoside,6,8-Dihydroxykaempferol, Isorhamnetin 3-O-galactoside, Quercetin3-O-rhamnosyl-rhamnosyl-glucoside, Kaempferol3-O-rhamnosyl-rhamnosyl-glucoside, Methylgalangin, Kaempferol 3,7,4′-O-triglucoside, 5,3′,4′-Trihydroxy-3-methoxy-6:7-methylenedioxyflavone,5,4′- Dihydroxy-3,3′-dimethoxy-6:7-methylenedioxyflavone, Jaceidin,Natsudaidain, 3-Methoxynobiletin, 3-Methoxysinensetin, Quercetin 3*-O-glucuronide, Quercetin 3′-sulfate, Quercetin 4′-O-glucuronide,Isorhamnetin 4′-O-glucuronide, Tamarixetin, Quercetin3-O-glucosyl-rutinoside Isoflavonoids Daidzein, Formononetin, Genistein,Biochanin A, Glycitein, Glycitin, 6″-O- Acetyldaidzin,6″-O-Malonylgenistin, Daidzin, Genistin, 6″-O- Acetylgenistin,6″-O-Acetylglycitin, 6″-O-Malonyldaidzin, 6″-O- Malonylglycitin,2′,7-Dihydroxy-4′,5′-dimethoxyisoflavone, 2-Dehydro-O-desmethylangolensin, 2′-Hydroxyformononetin,3′,4′,7-Trihydroxyisoflavan, 3′,4′,7-Trihydroxyisoflavanone,3′,7-Dihydroxyisoflavan, 3′- Hydroxydaidzein,3′-Hydroxy-O-desmethylangolensin, 4′,6,7- Trihydroxyisoflavanone,4′,7,8-Trihydroxyisoflavanone, 4′,7-Dihydroxy-3′- methoxyisoflavan,4′,7-Dihydroxy-6-methoxyisoflavan, 4-Hydroxyequol, 4′- O-Methylequol,5,6,7,3′,4′-Pentahydroxyisoflavone, 5,6,7,4′- Tetrahydroxyisoflavone,5,7,8,3′,4′-Pentahydroxyisoflavone, 5,7,8,4′- Tetrahydroxyisoflavone,5′-Hydroxy-O-desmethylangolensin, 5′-Methoxy-O- desmethylangolensin,6,7,3′,4′-Tetrahydroxyisoflavone, 6,7,4′- Trihydroxyisoflavone,6′-Hydroxyangolensin, 6′-Hydroxy-O- desmethylangolensin,7,3′,4′-Trihydroxy-6-methoxyisoflavone, 7,3′,4′- Trihydroxyisoflavone,7,8,3′,4′-Tetrahydroxyisoflavone, 7,8,4′- Trihydroxyisoflavone,Angolensin, Calycosin, Daidzein 4′,7-O- diglucuronide, Daidzein4′,7-disulfate, Daidzein 4′-O-glucuronide, Daidzein 4′-sulfate, Daidzein7-O-glucuronide, Dihydrobiochanin A, Dihydrodaidzein, Dihydrodaidzein7-O-glucuronide, Dihydroformononetin, Dihydrogenistein,Dihydroglycitein, Equol, Formononetin 7-O-glucuronide, Formononetin 7-sulfate, Genistein 4′,7-O-diglucuronide, Genistein 4′,7-disulfate,Genistein 4′- O-glucuronide, Genistein 4′-sulfate, Genistein5-O-glucuronide, Genistein 7- O-glucuronide, Genistein 7-sulfate,Glycitein 4′-O-glucuronide, Glycitein 7- O-glucuronide, Koparin,O-Desmethylangolensin, Orobol, Prunetin, Pseudobaptigenin, Puerarin,Daidzin 4′-O-glucuronide, Irisolidone 7-O- glucuronide, Tectorigenin7-sulfate, Tectorigenin 4′-sulfate, Irisolidone, Tectorigenin,Tectoridin, 5,7-Dihydroxy-8,4′-dimethoxyisoflavone, Isotectorigenin,Equol 7-O-glucuronide, Equol 4′-O-glucuronide, 8- Hydroxydaidzein,Daidzein 7-sulfate, Daidzein 4′-O-sulfo-7-O-glucuronide, Daidzein7-O-sulfo-4′-O-glucuronide, Equol 4′-sulfate, 3′,4′,5,7-Tetrahydroxyisoflavanone, 3′-O-Methylequol, 6-O-Methylequol, 3′-Hydroxygenistein, 3′-Hydroxydihydrodaidzein, 6-Hydroxydihydrodaidzein,3′-Hydroxyequol, cis-4-Hydroxyequol, 4′-Methoxy-2′,3,7-trihydroxyisoflavanone, Irilone, Vestitone, Sativanone, Butin, 3′-Hydroxymelanettin, Liquiritigenin, Melanettin, Stevenin, Violanone,Isoliquiritigenin, Dalbergin, 3′-O-Methylviolanone, 8-Hydroxydihydrodaidzein Lignans Secoisolariciresinol, Matairesinol,Lariciresinol, Pinoresinol, Syringaresinol, Isolariciresinol,Arctigenin, Trachelogenin, Medioresinol, 1- Acetoxypinoresinol,Secoisolariciresinol di-O-glucoside, Sesamin, Sesamolin, Sesamolinol,Sesaminol, Sesaminol 2′-O-b-D-glucosyl (1->2)-O- [b-D-glucosyl(1->6)]-b-D-glucoside, Sesaminol 2′-O-b-D-glucosyl (1->6)-O-b-D-glucoside, Sesaminol 2′-O-b-D-glucoside, Sesamol, Sesamolinol4′-O- b-D-glucosyl (1->6)-O-b-D-glucoside, 7-Hydroxymatairesinol,Isohydroxymatairesinol, Secoisolariciresinol-sesquilignan,Cyclolariciresinol, 7-Oxomatairesinol, Todolactol A, Conidendrin, 7-Hydroxysecoisolariciresinol, Nortrachelogenin,Lariciresinol-sesquilignan, Anhydro-secoisolariciresinol,Dimethylmatairesinol, Episesamin, Episesaminol, Sesaminol2′-O-b-D-glucosyl (1->2)-O-b-D-glucoside, Enterodiol, Enterolactone,Sesaminol 2-O-triglucoside, Schisandrin, Gomisin D, Schisandrol B,Tigloylgomicin H, Schisanhenol, Schisantherin A, Gomisin M2,Deoxyschisandrin, Schisandrin B, Schisandrin C, 2-Hydroxyenterodiol,4-Hydroxyenterodiol, 6-Hydroxyenterodiol, 2-Hydroxyenterolactone, 4-Hydroxyenterolactone, 6-Hydroxyenterolactone, 2′-Hydroxyenterolactone,4′- Hydroxyenterolactone, 6′-Hydroxyenterolactone,5-Hydroxyenterolactone, 7- Hydroxyenterolactone Non-phenolic4-Ethylbenzoic acid, Glycine, 1,3,5-Trimethoxybenzene, Vanilloylglycinemetabolites Alkylmethoxy- 4-Vinylguaiacol, 4-Ethylguaiacol,4-Vinylsyringol phenols Alkylphenols 5-Heneicosenylresorcinol,5-Heneicosylresorcinol, 5-Heptadecylresorcinol, 5-Nonadecenylresorcinol,5-Nonadecylresorcinol, 5-Pentacosenylresorcinol, 5-Pentacosylresorcinol,5-Pentadecylresorcinol, 5-Tricosenylresorcinol, 5- Tricosylresorcinol,Alk(en)ylresorcinols, total, Alkenylresorcinols, total,Alkylresorcinols, total, 3-Methylcatechol, 4-Methylcatechol,4-Ethylcatechol, 4-Vinylphenol, 4-Ethylphenol Betacyanins Betanin,Isobetanin, Betanidin, Isobetanidin Capsaicinoids Capsaicin CurcuminoidsCurcumin, Demethoxycurcumin, Bisdemethoxycurcumin Dihydro-Dihydrocapsaicin, Nordihydrocapsaicin capsaicins Furano- Bergapten,Psoralen, Xanthotoxin, Isopimpinellin, Angelicin coumarins Hydroxy-Syringaldehyde, Protocatechuic aldehyde, Vanillin,4-Hydroxybenzaldehyde, benzaldehydes Gallic aldehyde, p-Anisaldehyde,Ethyl vanillin, Vanillin 4-sulfate Hydroxy- 3-Methoxyacetophenone,2,3-Dihydroxy-1-guaiacylpropanone, Paeonol, 2,4- benzoketonesDihydroxyacetophenone 5-sulfate, 2-Hydroxy-4-methoxyacetophenone 5-sulfate, Resacetophenone, Norathyriol Hydroxycinnam- Ferulaldehyde,Sinapaldehyde aldehydes Hydroxy- Coumarin, Isocoumarin, Mellein,Scopoletin, Esculetin, Esculin, coumarins Umbelliferone,4-Hydroxycoumarin, Urolithin D, Urolithin B 3-sulfate, Urolithin A3,8-O-diglucuronide, Urolithin A 3,8-disulfate, Urolithin A, UrolithinB, Urolithin B 3-O-glucuronide, Urolithin C Hydroxyphenyl- Homovanillylalcohol alcohols Hydroxy- 2-Methoxy-5-prop-1-enylphenol, Anethole,Eugenol, Acetyl eugenol, [6]- phenylpropenes Gingerol, EstragoleMethoxyphenols Guaiacol, p-Anisidine Naphtoquinones Juglone,1,4-Naphtoquinone Phenolic Carnosic acid, Rosmanol, Carnosol,Epirosmanol, Rosmadial, Thymol, terpenes Carvacrol TyrosolsHydroxytyrosol, 3,4-DHPEA-AC, p-HPEA-AC, Oleuropein, Demethyloleuropein,3,4-DHPEA-EA, Ligstroside, 3,4-DHPEA-EDA, Hydroxytyrosol 4-O-glucoside,Oleoside dimethylester, Oleoside 11- methylester, Hydroxytyrosol1′-O-glucoside, p-HPEA-EDA, p-HPEA-EA, Oleuropein-aglycone,Ligstroside-aglycone, Elenolic acid, Tyrosol 4-O- glucuronide, Tyrosol4-sulfate, Hydroxytyrosol, total Other Coumestrol, Catechol, Pyrogallol,Phlorin, Phenol, Phloroglucinol, Arbutin, polyphenols Hydroquinone,3,4-Dihydroxyphenylglycol, 5,5′,6,6′-Tetrahydroxy-3,3′- biindolyl,Resorcinol, 1-Phenyl-6,7-dihydroxy-isochroman, 1-(3-methoxy-4-hydroxy)-phenyl-6,7-dihydroxy-isochroman, Lithospermic acid,Lithospermic acid B, Salvianolic acid B, Salvianolic acid C, Salvianolicacid D, Salvianolic acid G, Isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate Hydroxybenzoic Ellagic acid glucoside, Protocatechuicacid, Gallic acid, Vanillic acid, Ellagic acids acid, total, Gentisicacid, Ellagic acid, 4-Hydroxybenzoic acid, 3,4- Dimethoxybenzoic acid,Syringic acid, 5-O-Galloylquinic acid, Ellagic acid arabinoside, Ellagicacid acetyl-xyloside, Ellagic acid acetyl-arabinoside, 4- Methoxybenzoicacid, Gallic acid, total, Benzoic acid, 2-Hydroxybenzoic acid,3-Hydroxybenzoic acid, 2,3-Dihydroxybenzoic acid, 2,4- Dihydroxybenzoicacid, 1-O-Galloyl glucose, 4-Hydroxybenzoic acid 4-O- glucoside,Protocatechuic acid 4-O-glucoside, Gallic acid 4-O-glucoside, 3,5-Dihydroxybenzoic acid, 2,6-Dihydroxybenzoic acid, Gallic acid3-O-gallate, Gallic acid ethyl ester, Valoneic acid dilactone,2,6-Dimethoxybenzoic acid, 2-Hydroxy-4-methoxybenzoic acid, Sanguisorbicacid dilactone, Galloyl glucose, Lambertianin C, Sanguiin H-6, SanguiinH-10, Ellagitannins, total, Punicalagin, Gallagic acid, Tannic acid,Hydrolysable tannins, total, 3-O- Methylgallic acid, 4-O-Methylgallicacid, 3,4-O-Dimethylgallic acid, Punicalin, 4-Hydroxyhippuric acid,3-Hydroxyhippuric acid, 2- Hydroxyhippuric acid, Hippuric acid,Paeoniflorin, Vanillic acid 4-sulfate, 2,3,4-Trihydroxybenzoic acidHydroxy- p-Coumaric acid, 5-p-Coumaroylquinic acid, 4-p-Coumaroylquinicacid, cinnamic acids Caffeic acid, Feruloyl glucose, Ferulic acid,Caffeoyl tartaric acid, Rosmarinic acid, o-Coumaric acid, m-Coumaricacid, Sinapic acid, p- Coumaroyl glucose, p-Coumaroylquinic acid,3-Caffeoylquinic acid, Verbascoside, 4-Caffeoylquinic acid, p-Coumaroyltartaric acid, 2,5-di-S- Glutathionyl caftaric acid, Feruloyl tartaricacid, Caffeic acid ethyl ester, Cinnamoyl glucose, 5-Caffeoylquinicacid, 3-p-Coumaroylquinic acid, 2-S- Glutathionyl caftaric acid,5-Feruloylquinic acid, 4-Feruloylquinic acid, 3- Feruloylquinic acid,5-Sinapoylquinic acid, 4-Sinapoylquinic acid, 3- Sinapoylquinic acid,3,5-Dicaffeoylquinic acid, Isoferulic acid, Caffeoyl glucose, p-Coumaricacid 4-O-glucoside, Caffeic acid 4-O-glucoside, Ferulic acid4-O-glucoside, p-Coumaroyl tartaric acid glucosidic ester, p-Coumaricacid ethyl ester, Trans-Caffeoyl tartaric acid, Cis-Caffeoyl tartaricacid, Trans-p-Coumaroyl tartaric acid, Cis-p-Coumaroyl tartaric acid,Trans- Caffeic acid, Cis-Caffeic acid, Trans-p-Coumaric acid,Trans-Ferulic acid, Cis-p-Coumaric acid, Cis-Ferulic acid,3,4-Dimethoxycinnamic acid, Hydroxycaffeic acid, Caffeic acid, total,Sinapic acid, total, Chicoric acid, 5- 5′-Dehydrodiferulic acid,5-8′-Dehydrodiferulic acid, 1,2- Disinapoylgentiobiose,1-Sinapoyl-2-feruloylgentiobiose, 1,2- Diferuloylgentiobiose,1,2,2′-Trisinapoylgentiobiose, 1,2′-Disinapoyl-2- feruloylgentiobiose,1-Sinapoyl-2,2′-diferuloylgentiobiose, 1,2,2′- Triferuloylgentiobiose,8-O-4′-Dehydrodiferulic acid, 8-8′-Dehydrodiferulic acid,5-8′-Benzofuran dehydrodiferulic acid, Cis-3-Caffeoylquinic acid, 3,4-Dicaffeoylquinic acid, Cis-5-Caffeoylquinic acid, 3,4-Diferuloylquinicacid, 3,5-Diferuloylquinic acid, 1-Caffeoylquinic acid,1,3-Dicaffeoylquinic acid, 1,5-Dicaffeoylquinic acid,4,5-Dicaffeoylquinic acid, Dicaffeoylquinic acid,b-D-fructosyl-a-D-(6-O-(E))-feruloylglucoside, Avenanthramide 1p,Avenanthramide if, Avenanthramide 2p, Avenanthramide 2c, Avenanthramide2f, Avenanthramide lc, Avenanthramide is, Avenanthramide 2s, Sinapoylglucose, p-Coumaroyl malic acid, p-Coumaroyl glycolic acid,3-Caffeoyl-1,5-quinolactone, 4-Caffeoyl-1,5-quinolactone, Quinic acidesters, total, 3-Feruloyl-1,5-quinolactone, 4-Feruloyl-1,5-quinolactone, 3,4-Dicaffeoyl-1,5-quinolactone, 3-p-Coumaroyl-1,5-quinolactone, 4-p-Coumaroyl-1,5-quinolactone, Cinnamic acid, Caffeoyl 3-hydroxytyrosine, Caffeoyl aspartic acid, p-Coumaroyl aspartic acid, p-Coumaroyl tyrosine, Caffeoyl tyrosine, p-Coumaroyl 3-hydroxytyrosine,Isoverbascoside, Sinapine, Avenanthramide A2, Avenanthramide K,Campesteryl ferulate, Sitostanyl ferulate,4-O-8′,5′-5″-Dehydrotriferulic acid, 24-Methylcholestanol ferulate,24-Methylcholesterol ferulate, 24- Methyllathosterol ferulate,Stigmastanol ferulate, Sitosterol ferulate, Schottenol ferulate,24-Methylenecholestanol ferulate, Trans-5- Caffeoylquinic acid,Trans-3-Caffeoylquinic acid, 3-O-Methylrosmarinic acid, Sinapic acid4-O-glucuronide, Sinapic acid 4-sulfate, Feruloylglycine 4- sulfate,Feruloylglycine, Isoferulic acid 3-O-glucuronide, Isoferulic acid 3-sulfate, Ferulic acid 4-sulfate, Ferulic acid 4-O-glucuronide, Caffeicacid 4- sulfate, Caffeic acid 3-sulfate, p-Coumaric acid 4-sulfate,Feruloyl C1- glucuronide, Isoferuloyl Ci-glucuronide, Caffeic acid3-O-glucuronide, Caffeic acid 4-O-glucuronide, Caffeoyl C1-glucuronide,Chlorogenic acid, total, 1,5-Diferuloylquinic acid,1-Caffeoyl-5-feruloylquinic acid, 1-Feruloyl- 5-caffeoylquinic acidHydroxy- 3,4-Dihydroxyphenylacetic acid, 4-Hydroxyphenylacetic acid,Homovanillic phenylacetic acid, Homoveratric acid, Methoxyphenylaceticacid, 3-Hydroxyphenylacetic acids acid, 2-Hydroxyphenylacetic acid,4-Methoxyphenylacetic acid, Phenacetylglycine, Phenylacetic acid,4-Hydroxymandelic acid, 2-Hydroxy- 2-phenylacetic acid, Homovanillicacid 4-sulfate, 4-Hydroxyphenyllactic acid Hydroxy- Dihydro-p-coumaricacid, Dihydrocaffeic acid, 3,4-Dihydroxyphenyl-2- phenylpropanoicoxypropanoic acid, 3-Hydroxy-3-(3-hydroxyphenyl)propionic acid, 3-(3,4-acids Dihydroxyphenyl)-2-methoxypropionic acid, 3-Hydroxyphenylpropionicacid, Dihydroferulic acid 4-sulfate, Dihydroisoferulic acid3-O-glucuronide, Dihydrocaffeic acid 3-O-glucuronide, Dihydrocaffeicacid 3-sulfate, Dihydroferulic acid, Dihydroferulic acid4-O-glucuronide, Dihydrosinapic acid, Dihydroferuloylglycine 4-sulfate,Dihydroferuloylglycine, Danshensu, 3-Methoxy-4-hydroxyphenyllactic acid,3,4-Dihydroxyphenyllactic acid methyl ester, Hydroxydanshensu,3-Phenylpropionic acid, 3-Hydroxy-4- methoxyphenyllactic acid,Dihydroferulic acid 3-sulfate, 4-Hydroxyphenyl-2- propionic acidHydroxy- 5-(3′-Methoxy-4′-hydroxyphenyl)-γ-valerolactone,5-(3′-Methoxy-4′- phenylpentanoic hydroxyphenyl)-γ-valerolactone4′-O-glucuronide, 4-Hydroxy-(3′,4′- acids dihydroxyphenyl)valeric acid,5-(3′,4′-dihydroxyphenyl)-valeric acid, 5-(3′,4′,-dihydroxyphenyl)-γ-valerolactone,5-(3′,4′,5′-trihydroxyphenyl)-γ- valerolactone,5-(3′,5′-dihydroxyphenyl)-γ-valerolactone, 5-Hydroxyphenyl-γ-valerolactone, 3-Hydroxyphenylvaleric acid,5-(3′,5′-dihydroxyphenyl)-γ- valerolactone 3-O-glucuronide StilbenesTrans-Resveratrol, Trans-Resveratrol 3-O-glucoside, Piceatannol, Cis-Resveratrol, e-Viniferin, Pterostilbene, d-Viniferin, Cis-Resveratrol3-O- glucoside, Pallidol, Piceatannol 3-O-glucoside, Pinosylvin,Resveratrol 5-O- glucoside, Resveratrol, Resveratrol 3-O-glucoside,3,4,5,4′- Tetramethoxystilbene,3′-Hydroxy-3,4,5,4′-tetramethoxystilbene, 3-Hydroxy-4,5,4′-trimethoxystilbene, 4,4′-Dihydroxy-3,5-dimethoxystilbene, 4′-Hydroxy-3,4,5-trimethoxystilbene, 4-Hydroxy-3,5,4′-trimethoxystilbene,cis- Resveratrol 3-O-glucuronide, cis-Resveratrol 3-sulfate,cis-Resveratrol 4′-O- glucuronide, cis-Resveratrol 4′-sulfate,Resveratrol 3-O-glucuronide, Resveratrol 3-sulfate, Resveratrol4′-O-glucuronide, trans-Resveratrol 3,5- disulfate, trans-Resveratrol3,4′-disulfate, trans-Resveratrol 3-O-glucuronide, trans-Resveratrol3-sulfate, trans-Resveratrol 4′-O-glucuronide, trans- Resveratrol4′-sulfate, Dihydroresveratrol

EQUIVALENTS AND SCOPE

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Because such embodimentsare deemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the invention can be excluded from any claim,for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,Figures, or Examples but rather is as set forth in the appended claims.Those of ordinary skill in the art will appreciate that various changesand modifications to this description may be made without departing fromthe spirit or scope of the present invention, as defined in thefollowing claims.

We claim:
 1. A method of treating a human subject having a disease ordisorder associated with elevated levels of trimethylamine N-oxide(TMAO) comprising administering to the subject a pharmaceuticalcomposition comprising a glycan therapeutic preparation in an amounteffective to treat the disease or disorder, wherein (i) the glycantherapeutic preparation comprises branched glycans that compriseglucose, galactose, arabinose, mannose, fructose, xylose, fucose, orrhamnose glycan units; (ii) the average degree of branching (DB) of thebranched glycans in the glycan therapeutic preparation is between 0.01and 0.5; (iii) at least 50% of the glycans in the glycan therapeuticpreparation have a degree of polymerization (DP) of at least 3 and lessthan 30 glycan units; (iv) the ratio of alpha- to beta-glycosidic bondspresent in the glycans of the glycan therapeutic preparation is betweenabout 0.8:1 to about 5:1; (v) at least two of the glycosidic bondsindependently comprise a 1→2 glycosidic bond, a 1→3 glycosidic bond, a1→4 glycosidic bond, or a 1→6 glycosidic bond; and (vi) the glycantherapeutic preparation has a final solubility limit in water of atleast about 60 Brix at 23° C.
 2. The method of claim 1, wherein thedisease or disorder associated with elevated levels of TMAO is chronickidney disease (CKD).
 3. The method of claim 1, wherein the average DPof the glycan therapeutic preparation is between about DP6 and aboutDP12.
 4. The method of claim 1, wherein at least 60% of the glycantherapeutic preparation has an average molecular weight of less than2000 g/mol.
 5. The method of claim 1, wherein the average degree ofbranching (DB) of the branched glycans in the glycan therapeuticpreparation is between 0.05 and 0.25.
 6. The method of claim 1, whereinthe glycan therapeutic preparation comprises branched glycans consistingof glucose glycan units.
 7. The method of claim 1, wherein the glycantherapeutic preparation comprises branched glycans consisting of glucoseand mannose glycan units.
 8. The method of claim 1, wherein thepharmaceutical composition is administered daily.
 9. The method of claim1, wherein the method further comprises administering a second drug orpharmaceutical composition.
 10. The method of claim 1, wherein theglycan therapeutic preparation comprises branched glycans comprisingglucose glycan units.
 11. The method of claim 1, wherein the glycantherapeutic preparation comprises branched glycans comprising glucoseand mannose glycan units.